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Activation, Desensitization and Potentiation of Alpha7 Nicotinic Acetylcholine Receptors

Permanent Link: http://ufdc.ufl.edu/UFE0044048/00001

Material Information

Title: Activation, Desensitization and Potentiation of Alpha7 Nicotinic Acetylcholine Receptors Relevance to Alpha7-Targeted Therapeutics
Physical Description: 1 online resource (230 p.)
Language: english
Creator: Williams, Dustin K
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: cytotoxicity -- electrophysiology -- patch-clamp -- potentiation -- receptor -- single-channel
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are recognized therapeutic targets for cognitive and neurodegenerative disorders. While activation of the ion channel is primarily controlled by the agonist binding sites, it can also be regulated in positive or negative ways by the binding of ligands to other modulatory sites. The functional significance of two highly conserved tryptophan residues located in agonist binding sites are investigated in homomeric alpha7 and heteromeric alpha4beta2 receptors. The data suggest that tryptophan 149 is critical for activation by diverse agonists, but the tryptophan at position 55 may be important for regulating the ability of benzylidene anabaseine compounds to selectively activate the alpha7 receptor. A method of conditionally eliminating agonist binding sites is used to investigate the functional significance of multiple binding sites in heteromeric muscle-type and alpha7 receptors. The results suggest that nAChRs are capable of being effectively activated under conditions of submaximal agonist occupancy. Characterization of the alpha7 positive allosteric modulator PNU-120596 reveals the existence of two distinct alpha7 desensitized states that are distinguished by their stability in the presence of PNU-120596. Desensitized states that are stable in the presence of PNU-120596 are induced by conditions promoting strong ion channel activation and influenced by both agonist and modulator concentrations. Outside-out patch clamp recordings illustrate that PNU-120596 has a profound effect on currents from individual alpha7 channels. A novel cell line was developed that stably expresses functional human alpha7 receptors. This cell line was used to investigate the in vitro cytotoxicity profile of PNU-120596. In addition, the cell line was used to evaluate the temperature dependence of PNU-120596 potentiating activity that was recently reported. The findings suggest that PNU-120596 can produce concentration-dependent cytotoxic effects and confirm that PNU-120596 potentiation is reduced at physiological temperatures. However, some endogenous factors, such as serum albumins, may partially preserve the ability of PNU-120596 to potentiate alpha7-mediated responses at 37°C. The work presented in this dissertation will hopefully aid the rational design of therapeutics targeted to the alpha7 nAChR.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Dustin K Williams.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Papke, Roger L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044048:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044048/00001

Material Information

Title: Activation, Desensitization and Potentiation of Alpha7 Nicotinic Acetylcholine Receptors Relevance to Alpha7-Targeted Therapeutics
Physical Description: 1 online resource (230 p.)
Language: english
Creator: Williams, Dustin K
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: cytotoxicity -- electrophysiology -- patch-clamp -- potentiation -- receptor -- single-channel
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are recognized therapeutic targets for cognitive and neurodegenerative disorders. While activation of the ion channel is primarily controlled by the agonist binding sites, it can also be regulated in positive or negative ways by the binding of ligands to other modulatory sites. The functional significance of two highly conserved tryptophan residues located in agonist binding sites are investigated in homomeric alpha7 and heteromeric alpha4beta2 receptors. The data suggest that tryptophan 149 is critical for activation by diverse agonists, but the tryptophan at position 55 may be important for regulating the ability of benzylidene anabaseine compounds to selectively activate the alpha7 receptor. A method of conditionally eliminating agonist binding sites is used to investigate the functional significance of multiple binding sites in heteromeric muscle-type and alpha7 receptors. The results suggest that nAChRs are capable of being effectively activated under conditions of submaximal agonist occupancy. Characterization of the alpha7 positive allosteric modulator PNU-120596 reveals the existence of two distinct alpha7 desensitized states that are distinguished by their stability in the presence of PNU-120596. Desensitized states that are stable in the presence of PNU-120596 are induced by conditions promoting strong ion channel activation and influenced by both agonist and modulator concentrations. Outside-out patch clamp recordings illustrate that PNU-120596 has a profound effect on currents from individual alpha7 channels. A novel cell line was developed that stably expresses functional human alpha7 receptors. This cell line was used to investigate the in vitro cytotoxicity profile of PNU-120596. In addition, the cell line was used to evaluate the temperature dependence of PNU-120596 potentiating activity that was recently reported. The findings suggest that PNU-120596 can produce concentration-dependent cytotoxic effects and confirm that PNU-120596 potentiation is reduced at physiological temperatures. However, some endogenous factors, such as serum albumins, may partially preserve the ability of PNU-120596 to potentiate alpha7-mediated responses at 37°C. The work presented in this dissertation will hopefully aid the rational design of therapeutics targeted to the alpha7 nAChR.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Dustin K Williams.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Papke, Roger L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044048:00001


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1 ACTIVATION, DESENSITIZATION AND POTENTIATION OF ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTORS: RELEVANCE TO ALPHA7 TARGETED THERAPEUTICS By DUSTIN KYLE WILLIAMS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Dustin Kyle Williams

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3 To the most important people in my life: my family

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4 ACKNOWLEDGMENTS I would like to thank my wife, Julie Williams, and my parents, Kevin and Kathy Williams for their unconditional love and flawless support through the graduate school experience, even when I have had to place work before them. I would like to thank Dr. Roger Papke for his mento rship over the last five years. I have had more success under his arm than I ever could have expected when I started graduate school. In addition, I am thankful for the many collaborative meetings I participated in with Dr. Nicole Horenstein and Dr. Jin gyi Wang. I would like to thank Mathew Kimbrell for his fine work in helping to create the stably transfected cell line s and for the interactions I have had with all members of the Papke laboratory. I wish to express gratitude to the members of my advis ory committee, Dr. Nicole Horenstein, Dr. Brian Cooper, Dr. Michael King, and Dr. Brian Law, for their counsel and guidance. I am thankful for the financial supp ort I have received from the National Institute on Aging Training Grant T32 AG000196.

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5 TABLE OF CONTENTS P age ACKNOWLEDGMENTS ................................ ................................ ................................ ... 4 LIST OF TABLES ................................ ................................ ................................ ............. 8 LIST OF FIGURES ................................ ................................ ................................ ........... 9 LIST OF ABBREVIATIONS ................................ ................................ ............................ 12 ABSTRACT ................................ ................................ ................................ .................... 14 CHAPTER 1 ANIMAL ELECTRICITY: HISTORICAL INTRODUCTION ................................ ..... 16 Pre Galvani to Hod g kin & Huxley ................................ ................................ ............ 16 Chemical Neurotransmission, Receptors, and Ion Channels ................................ .. 28 2 NICOTINIC ACETYLCHOLINE RECEPTORS: INTRODUCTION ......................... 37 The Cys Loop Superfamily of Ligand Gated Ion Channels ................................ .... 37 nAChRs of muscle and electric organs ................................ ................................ .. 38 General Features of nAChR Structure Learned from Muscle type nAChRs .......... 38 Neuronal nAChR Su bunits ................................ ................................ ..................... 41 Physiology, Expression, and Functional Roles of Muscle type nAChRs ................ 43 Physiology, Expression, and Functional Roles of Neuronal nAChRs ..................... 44 General Distinguishing Features of Neuronal nAChRs ................................ ..... 44 Peripheral Neuronal nAChRs ................................ ................................ ............ 45 Central Neuronal nAChRs ................................ ................................ ................ 45 High Affinity Binding Sites ................................ ................................ ................. 49 Low Affinity Binding Sites: 7 nAChRs ................................ ................................ 51 Distinguishing Functional Characteristics of the homomeric 7 nAChR ........... 51 Implications of 7 nAChR in Pathophysiology ................................ .................. 54 Therapeutic Targe ting of the 7 nAChR through Positive Allosteric Modulation ................................ ................................ ................................ ......... 57 3 METHODS ................................ ................................ ................................ .............. 65 !!! cDNA Clones ................................ ................................ ................................ ........... 65 Chemicals ................................ ................................ ................................ ................ 65 Si te Directed Mutations ................................ ................................ ........................... 66 Preparation of RNA for Injections into Xenopus laevis Oocytes .............................. 66 Expression in Xenopus laevis Oocytes ................................ ................................ ... 66 Two Electrode Voltage Clamp Electrophysiology ................................ .................... 67 Transient Transfection of BOSC23 Cells ................................ ................................ 69 Generation of HEK293 Cells Stably Expressing Human 7 and Human ric3 ......... 70 Immunoprecipitation and Western Blot ................................ ................................ .... 72 Fluorescence Microscopy ................................ ................................ ........................ 73 Equillibrium Radioligand Binding Assay ................................ ................................ .. 73 Cytotoxicity Experime nts ................................ ................................ ......................... 74

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6 Outside out Patch Clamp Electrophysiology ................................ ........................... 75 !!!! Whole c ell Patch Clamp Electrophysiol ogy ................................ ............................. 79 4 DIFFERENTIAL REGULATION OF RECEPTOR ACTIVATION AND AGONIST SELECTIVITY BY HI GHLY CONSERVED TRYPTOPHANS IN THE NICOTINIC ACETYLCHOLINE RECEPTOR BINDING SITE ................................ .................... 83 Introduction ................................ ................................ ................................ ............. 83 Results ................................ ................................ ................................ .................... 85 Mutation of W55 or W57 of 7 and 4 2 Receptors, respectively, A lters the Pharmacology and Regulates the S electivity of 4OH GTS 21. ......................... 85 Absolute Efficacy of ACh ................................ ................................ ................... 86 Relative Effic acy of 7 selective Agonis ts Compared with ACh ........................ 87 M utation of W149 in B oth 7 and 4 2 Receptors Reduced Receptor A ctivation by B oth ACh and 7 Selective A gonists. ................................ ........................... 90 Discussion ................................ ................................ ................................ ............... 91 5 THE EFFECTIVE OPENING OF NICOTINIC ACETYLCHOLINE RECEPTORS WITH SINGLE AGONIST BINDING SITES ................................ .......................... 10 4 Introduction ................................ ................................ ................................ ........... 10 4 Results ................................ ................................ ................................ .................. 105 Identification of the 7L119C Mutation as a Tool to Stud y nAChR B inding S ites ................................ ................................ ................................ ................. 106 Effects of 7 L119C Ratios in M ixed 7 Wild Type/Mutant H eteromers ........... 106 Effects of ACh I nse nsitive Mutant Ratios in M ixed 7 Wild Type/M utant H etero mers ................................ ................................ ................................ ...... 108 Effe cts of Mutations H omologous to 7L119C in N on Subunits of Muscle Type R eceptors ................................ ................................ ................................ 109 Discussion ................................ ................................ ................................ ............. 115 6 INVESTIGATION OF THE MOLE CULAR MECHANISM OF THE ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR POSITIVE ALLOSTERIC MODULATOR PNU 120596 PROVIDES EVIDENCE FOR TWO DISTINCT DESENSITIZED STATES ................................ ................................ ................... 1 30 Introduction ................................ ................................ ................................ ........... 130 Results ................................ ................................ ................................ ................... 131 ACh Evoked R esp onses of 7 nAChR E xpressed in Xenopus O ocytes ......... 131 ACh Evoked R esponses of 7 nAChR Expressed in BOSC2 3 C ells in the Absence and P resence of PNU 120596 ................................ .......................... 132 Factors Limiting the Potentiati ng E ffects of PNU 120596 on 7 M ediated C urrents ................................ ................................ ................................ ........... 134 Single Channel B ursts of 7 nAChR P romoted by PNU 120596 .................... 136 Discussion ................................ ................................ ................................ ............. 141

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7 7 A NOVEL CELL LINE STABLY EXPRESSING HUMAN ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTORS REVEALS THAT PNU 120596 POTENTIATION AND CYTOTOXICITY ARE ATTENUATED AT PHYSIOLOGICAL TEMPERATURE ................................ ................................ ................................ ... 15 5 Introduction ................................ ................................ ................................ ............ 155 Results ................................ ................................ ................................ ................... 157 Expression of hric3 and h 7 mRNA in Hygromycin and G418 R esistant C lones ................................ ................................ ................................ .............. 157 I dentification of the h 7 Protein via Western Blot in Antibiotic Resistant C lones ................................ ................................ ................................ .............. 157 Labeling of HEK h 7/hric3 C ell s with Alexa488 C onjugated B ungarotoxin 157 Specific Binding of [ 125 I] Bungarotoxin to I ntact HEK h 7/hric3 C ells .......... 15 8 ACh Evoked R esponses from HEK h 7/hric3 Cells and I nhibition of those Currents wit h MLA in a Concentration D ependent manner ............................. 159 In Vitro Cytotoxicity Profile of PNU 120596 in HEK h 7/hric3 C ells ................ 161 Bovine Serum Albumin Eliminates the Temperature Dependence of PNU 120596 Toxicity and, to a Lesser Degree, Potentiation Activity ....................... 164 The Temperature Dependence of PNU 120596 is Confirmed through Whole Cell Patch Clamp Recordings ................................ ................................ .......... 166 Discuss ion ................................ ................................ ................................ ............. 169 8 SUMMARY AND CONCLUSIONS ................................ ................................ ........ 1 8 5 REFERENCES ................................ ................................ ................................ ............. 19 2 BIOGRAPHICAL SKETCH ................................ ................................ ........................... 2 30

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8 LIST OF TABLES Table page 4 1 EC 50 and I max values of ACh, Choline, 4OH GTS 21, and AR R17779 in wild type and mutant 7 and 4 2 receptors. ................................ .......................... 103 5 1 7 L119C ratio experiments net charge data after MTSEA treatment ......... ....... 12 9 5 2 MTSEA effects on muscle mutants expressed in Xenopus oocytes !!! !! 12 9 5 3 Peak current and NP open measurements from the outside out patch clamp Experiments !!!!!!!!!!!!!!!!!!!!!!!!!!!! ..12 9 5 4 Fit time constants from the burst duration histograms !!!!!!!!!! ... 12 9 6 1 Fit time con stants from event duration hist ograms in the presence of 300 M ACh and 10 M PNU 120596 !!!!!!!!!!!!!!!! !!! ..15 4 7 1 10 90% rise times and rise slopes with increasing concentrations of ACh !!!!!! !!!!!! .. !!!!!!!!!!!!!!!!!!! ..18 4

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9 LIST OF FIGURES Figure page 4 1 Multiple sequence alignment and hypothetical l ocalization of 4W154, 2W57, 7W55, and 7W149. ................................ ................................ .......... 96 4 2 Concentration response relationships of wild type 7 and 4 2 receptors to ACh, choline, 4OH GTS 21, and AR R17779. ................................ .............. 97 4 3 Functional responses of human 7W55 and human 4 2W5 7 mutant receptors relative to ACh induced maximum responses in wild type. ................. 98 4 4 Concentration response relationship of 7W55 mutant receptors to ACh, choline, 4OH GTS 21, and AR R17779.. ................................ ............................ 99 4 5 Concentration response relationships of 4 2W57 mutants to ACh, cho line, 4OH GTS 21, and AR R17779 ................................ ................................ ........ 100 4 6 Concentration response relationships of 7W149 mutants to ACh, cho line, 4OH GTS 21, and AR R17779 ................................ ................................ ........ 101 4 7 Concentration response relationships of 4W154 2 mutants to ACh, choline, 4OH GTS 21, and AR R17779 ................................ ................................ ......... 102 5 1 Location of the 7L119 residue and the effect of MTSEA on L119C mutant receptors ................................ ................................ ................................ ........... 122 5 2 Co Expres sion of either L119C or Y188F with wild type 7 subunits at varying ratios ................................ ................................ ................................ .... 123 5 3 Probing the 7Y188F mutant receptor with selective and non selective agonists. ................................ ................................ ................................ ............ 124 5 4 The effect of MTSEA on mu scle type receptors with mutations homologous to 7 L119C in muscle # $ and % subunits ................................ ........................ 125 5 5 ACh concentration response data for muscle type single subunit mutants before and after MTSEA treatment. ................................ ................................ .. 126 5 6 The effect of MTSEA treatment on peak and NP open responses from recept ors expressed in BOSC 23 cells ................................ .............................. 127 5 7 Single channel traces and fit burst duration histograms from wild type 1 1 $# and 1 1 $# L121C receptors before and af ter MTSEA treatment as indicated. 12 8

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10 6 1 The basic characterization of 7 macroscopic currents, comparison to currents from heteromeric 4 2 nAChR, and the effects of PNU 120596. ....... 146 6 2 The low intrinsic P open of 7 is enhanced by PNU 120596. ............................... 147 6 3 Factors defining and limiting the potentiating effects of PNU 120596 on 7 nAChR expressed in Xenopus oocytes ................................ ............................ 148 6 4 Agonis t concentration dependence on the onset and decline of potentiation by PNU 120596 in outside out patches containing 7 receptors ..................... 149 6 5 Activity dependence of PNU 120596 potentiation onset and decline ............... 150 6 6 Recovery from PNU 1205 96 insensitive desensitization. ................................ 151 6 7 Single channel 7 bursts evoked by 300 M ACh and potentiated by 10 M PNU 120596. ................................ ................................ ................................ ..... 152 6 8 Single channel 7 bursts ev oked by 300 M ACh and potentiated by 10 M PNU 120596 persist despite the removal of AC h and in a MLA sensitive manner ................................ ................................ ................................ ............. 153 7 1 Expression of human 7 and ric3 by the HEK h 7/hric3 cell line. .................... 173 7 2 Labeling of intact HEK h 7/hric3 cells with Alexa Fluor488 bungarotoxin. ... 174 7 3 Saturation binding of [ 125 I] bungarotoxin binding to intact HEK h 7/hric3 cells ................................ ................................ ................................ .................. 175 7 4 ACh concentration response relationship and inhibition of currents by MLA from HEK h 7/hric3 cells. ................................ ................................ ................. 176 7 5 Temperature dependence of PNU 120596 cytotoxicity in HBSS solutions ...... 177 7 6 Sensitivity of the cytotoxic effect of 100 M choline + 10 M PNU 120596 treatment in HBSS at 28 ¡ C to the competitive antagonist MLA. ....................... 178 7 7 Elimination of the temperature depend ence of PNU 120596 cytotoxicity ........ 179 7 8 Sens itivity of the cytotoxic effect of 100 M choline + 10 M PNU 120596 treatment in HBSS with 30 M BSA at 37 ¡ C to the competitive antagonist MLA and the non competitive antagonist memamylamine. ............................... 180 7 9 Controls for the whole ce ll patch clamp recordings illustrating the temperature dependence of PNU 120596 potentiation. ................................ .... 181 7 10 Temperature dependence of PNU 120596 potentiation of 7 mediated responses. ................................ ................................ ................................ ......... 182

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11 7 11 Modest p reservation of PNU 120596 potentiation at 37 ¡ C in solutions containing 30 M BSA ................................ ................................ ...................... 183 8 1 Proposed qualitative models for the activation, desensitization, and modulation of 7 nAChR ................................ ................................ .................. 191

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12 LI ST OF ABBREVIATIONS 4OH GTS 21 3 (4 hydroxy 2 methoxybenzylidene) anabaseine 5 HI 5 hydroxyindole A 867744 4 (5 (4 chlorophenyl) 2 methyl 3 propionyl 1 H pyrrol 1 yl) benzenesulfonamide AChBP acetylcholine binding protein ACh acetylcholine AR R17779 ( ) S piro [ 1 azabicyclo(2.2 .2)octane 3,5 oxazolidin 2 one] BSA bovine serum albumin CCMI N (4 chlorophenyl) alpha [[(4 chloro phenyl)amino]methylene] 3 methyl 5 isox azoleacet amide D i PNU 120596 insensitive desensitization DMEM Dulbecco's modified e agl e m edium DPBS Dulbecco's phosphate buffered saline D s PNU 120596 sensitive desensitization ERK1/2 extracellular signal related kinase 1 and 2 FBS fetal bovine serum GTS 21 3 (2,4 dimethoxybenzylidene) anabaseine HBSS Hank's balanced saline solution HEK293 human embryonic kidney 293 cell line I max maximal current JNJ 1930942 2 [[4 fluoro 3 (trifluoromethyl)phenyl]amino] 4 (4 pyridinyl) 5 thiazolemethanol LBD ligand binding domain LY 2087101 [2 [(4 Fluorophenyl)amino] 4 methyl 5 thiazolyl] 3 thienylmethanone

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13 MLA methyllycaconitine MTSEA 2 aminoethyl methanethiosulfonate MTSET [2 (trimethylammonium)ethyl] methanethiosulfonate N number of ion channels in a patch nAChR nicotinic acetylcho line receptor NP open Absolute probability of channel opening NS 1738 1 (5 chloro 2 hydroxy phenyl) 3 (2 chloro 5 trifluoromethyl phenyl) urea O* isolated, short lived channel opening O' relatively long lived channel opening that primarily occurs in g roups or "bursts" PAM positive allosteric modulator PNU 120596 1 (5 chloro 2,4 dimethoxy phenyl) 3 (5 methyl isoxazol 3 yl) urea PNU 282987 N (3 R ) 1 Azabicyclo[2.2.2]oct 3 yl 4 chlorobenzamide P open probability of channel opening RT PCR reverse transcriptase polymerase chain reaction SB 206553 3,5 dihydro 5 methyl N 3 pyridinylbenzo[1,2 b:4,5 b']di pyrrole 1(2H) carboxamide SEM standard error of the mean t crit critical duration; burst delimiter value TQS 3a,4,5,9b Tetrahydro 4 (1 naphtha lenyl) 3 H cyclopentan[ c ]quinoline 8 sulfonamide

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ACTIVATION, DESENSIT IZATION AND POTENTIATION OF ALPHA7 NICOTINIC ACETYLCHOLINE RECEPT ORS: RELEVANCE TO ALPHA7 TARGE TED THERAPEUTICS By Dustin Kyle Williams May 2012 Chair: Roger L. Papke Major: Medical Sciences Neuronal nicotinic acetylcholine receptors (nAChRs) are recog nized therapeutic targets for cognitive and neurodegenerative disorders While activation of the ion channel is primarily controlled by the agonist binding sites, it can also be regulated in positive or negative ways by the binding of ligands to other mod ulatory sites. The functional significance of two highly conserved tryptophan residues located in agonist binding sites are investigated in homomeric 7 and heteromeric 4 2 receptors. The data suggest that tryptophan 149 is critical for activation by di verse agonists, but the tryptophan at position 55 may be important for regulating the ability of benzylidene anabaseine compounds to selectively activate the 7 receptor. A method of conditionally eliminating agonist binding sites is used to investigate t he functional significance of multiple binding sites in heteromeric muscle type and 7 receptors. The results suggest that nAChRs are capable of being effectively activated under conditions of submaximal agonist occupancy. Characterization of the 7 posi tive allosteric modulator PNU 120596 reveals the existence of two distinct 7 desensitized states that are distinguished by their stability in the presence of PNU 120596. Desensitized states

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15 that are stable in the presence of PNU 120596 are induced by con ditions promoting strong ion channel activation and influenced by both agonist and modulator concentrations. Outside out patch clamp recordings illustrate that PNU 120596 has a profound effect on currents from individual 7 channels. A novel cell line wa s developed that stably expresses functional human 7 receptors. This cell line was used to investigate the in vitro cytotoxicity profile of PNU 120596. In addition, the cell line was used to evaluate the temperature dependence of PNU 120596 potentiating activity that was recently reported. The findings suggest that PNU 120596 can produce concentration dependent cytotoxic effects and confirm that PNU 120596 potentiation is reduced at physiological temperatures. However, some endogenous factors such as serum albumins, may partially preserve the ability of PNU 120596 to potentiate 7 mediated responses at 37¡C The work presented in this dissertation will hopefully aid t he rational design of therapeutics targeted to the 7 n AChR.

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16 CHAPTER 1 ANIMAL ELECTRICITY: HISTORICAL INTRODUCT ION For many centuries the prevailing theories of neurotransmission had no concept of electricity. "Animal spirits", an immeasurable force thought to be the source of animation, imagination, reason, and memory, was believed to be contained within the fluid stored in the ventricles and distributed through out the body via the nerves which acted as conduits of this vital fluid [ 1 2 ] This fundamental hypot hesis was upheld and propagated for more than 1,500 years by many well known and influential philosophers, physicians, and scientists including Aristotle, Galen, Descartes, Borelli, and Fontanta, each contributing unique variations to the basic idea [ 2 4 ] It was not until the late 18th century that Luigi Galvani discovered electricity as the currency of the nervous system, and even then this important discov ery was not readily accepted. Understanding bioelectricity as we do today was truly a multi national effort that took place over hundreds of years. Pre Galvani to Hod g kin & Huxley By the 1660s, Jan Swammerdam (The Netherlands; 1637 1680) had shown that mu scles could contract without any physical connection to the brain, providing perhaps the first e vidence against the fallacious animal spirits hypothesis [ 4 ] He devised an isolated nerve muscle prepa ration from the frog and showed that mechanical stimulation of the nerve resulted in muscle contraction. His experiments also showed that muscles did not increase in volume (ie by an influx of "animal spirits") during contraction [ 3 4 ] He concluded that A simple and natural motion or irritation of the ner ve alone is necessary to produce muscular motion, whether it has its origin in the brain, or in the marrow, or elsewhere" [ 5 ] However, Swammerdam's work was unable

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17 to convince the scientific community at large that nervous system function occurred by a mechanism other than animal spirits [ 4 ] In the mid 1700s, Albrecht von Haller (Switzerland; 1708 1777) promoted his "doctrine of irritability", which was influenced by the work of Swammerdam and Francis Glisson (England; 1599 1 677), suggesting that external forces or irritations elicit responses from tissues that are dependent on the tissue's internal organization [ 4 6 7 ] Aware of von Haller's concept of irritability, Luigi Galvani (Italy; 1737 1798) and his assistants made an observation of profound scientific importance that set into motion the events that changed the definition of "animal spirits" to a physically understandable and measurable force underlying the function of the nervous system. While some claim that Luigi Galvani made his discovery by chance [ 4 ] it seems that Galvani sought to test the specific hypothesis that electricity was a source of "irritation" that could evoke muscle contraction based on his experimental set up and preparation at the time his discovery was made [ 6 7 ] In Galvani's time, elect ricity was still poorly understood, often thought of as a mystical force and often the subject of amusement and speculation. It was well known that frictional or static electricity could be produced by rubbing amber, glass, or rubber. Electrostatic gener ators, which operate on this principle, had been invented prior to Galvani's birth in 1737 [ 3 8 ] In 17 46 Pieter van Musschenbroek (The Netherlands; 1692 1761) developed a method for storing electricity produced with electrostatic generators, which could later be used at will, in a type of capacitor known as a Leyden jar [ 3 ] With these two inventions and his own version of Swammerdam's frog nerve muscle pr eparation consisting of the frog's hind limbs and exposed crural nerves [ 5 7 ] the discovery was made in 1781 when one of Galvani's assistants, probably either his wife Lucia Galeazzi

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18 Galvani or nephew Giovanni Aldini, touched the exposed nerve with a surgical instrument at the same time that a nearby frictional electricity generator sparked; the nerves h ad been stimulated by electrostatic induction and the muscles contracted as if the frog legs were alive [ 7 ] Throughout 10 years of experimentation Galvani performed numerous experiments in which he noted a non linear relationship between stimulus intensity of force of muscle co ntraction, a refractory period, and muscle contractions evoked by atmospheric electricity. Galvani came to the conclusion that electricity is intrinsic to animal tissue, hypothesizing that a state of electrical disequilibrium exists in muscle and that mus cle stores this energy in a way comparable to a Leyden jar [ 1 5 7 ] Galvani's ideas pre dated any und erstanding or demonstration of ions, membranes, and ion channels, but laid the foundation for their discovery. Galvani's findings quickly became well known throughout Europe; however, they were not immediately accepted and even faced considerable adversit y from the physicist Alessandro Volta (Italy; 1745 1827). Volta realized that small currents are produced when two different metals come in contact and this observation led him to question the concept of "animal electricity" based on a small sampling of G alvani's experiments: those in which muscle contractions were observed only if a metallic arc connecting the nerve and muscle was bimetallic and others in which contractions occurred when the preparation was hung on brass hooks, which were suspended from iron gratings, in the absence of atmospheric electricity or other external stimulation. Volta argued that electricity was not produced or stored in animal tissue, but rather that the muscle merely acted as a sensitive detector of electricity produced unkno wingly through contact of dissimilar conductors [ 1 6 ] Although Volta correctly realized that

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19 contact o f dissimilar metals could produce an external stimulus sufficient to evoke muscle contraction, he failed to acknowledge Galvani's demonstrations that muscle contractions occur in the complete absence of conducting metals. He also failed to acknowledge the independent confirmation and extension of Galvani's findings to birds, mammals, and reptiles by Alexander von Humboldt (Germany; 1769 1859) [ 8 9 ] In 1800, Volta's argument became widely accepted when he invented the first battery, the voltaic pile, which was essentially a stack of alternating copper, and zinc discs separated by saline soaked paper [ 1 ] The voltaic pile was capable o f generating a sustained direct current for the first time and when connected to the nerves of a frog leg preparation could evoke muscle contraction. In contrast to Volta, who received great honor and respect for his finding, Galvani lost his position at the University of Bologna and Academy of Sciences when he refused to make an oath of allegiance to Napoleo n in the late 1790s just prior to his death [ 7 ] Though not widely accepted scientifically, Galvanism had an important cultural impact throughout much of Europe. The public demonstrations by Giovanni Aldini in which he applied electricity to the brains of recently executed criminals, suggested to many that electricity could revive the dead. In addition, Galvani's discovery provided the inspiration for Mary Shelly's novel "F rankenstein" [ 7 10 ] At the time, Volta succeeded in convincing the majority of the scientific world that he was correct, however, his very discovery initiated the physical studies of electricity that led to improved equipment and an acceptance of bioelectricity during the next 50 years. In 1820, Hans Oersted (Denmark; 1777 1851) discovered that current movin g through a wire deflected the needle of a magnetic compass. The phenomenon of

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20 electromagnetism was soon applied with the invention of the first ammeter, later known as the galvanometer in honor of Galvani, that same year by Johann Schweigger (Germany; 17 79 1857) [ 10 ] Leopoldo N obili (Italy; 1784 1835) created the astatic galvanometer, a refinement of Schweigger's galvanometer, which effectively cancels the Earth's magnetic field allowing for greater instrument sensitivity [ 10 ] Nobili was the first to measure current flow through Galvani's frog prepara tion in 1828 with his instrument, but he seemed more interested in developing the sensitivity of his galvanometer than in studying the "intrinsic frog current", which he attributed to a thermoelectric effect [ 7 10 ] In the late 1830s and early 1840s, Carlo Matteucci (Italy; 1811 1868) made several important observations that helped establish the validity of bi oelectricity. Matteucci discovered that "injury currents" flow, and could be measured with a Nobili galvanometer, between intact and cut surfaces of denervated muscle. He then stacked several muscles on top of each other, with the injured surface of one muscle in contact with the intact surface of another, and showed that the measured currents were proportional to the number of muscles or elements in series, similar to the voltaic pile [ 1 10 ] In addition, Matteucci demonstrated that current stops flowing between injured and intact surfaces of muscle upon strychnine induced tetanus, providing perhaps the first electrophys iological evidence that pharmacological treatment with biologically active compounds manipulates measurable bioelectricity. Further, Matteucci discovered the phenomenon of "induced twitch", in which the contraction of one muscle produces an electrical dis charge that is sufficient to induce contraction of another muscle when the only contact between the two muscles is a sciatic nerve [ 7 ]

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21 Emil du Bois Reymond (Germany; 1818 1896) became interested in electrophysiology during the early 1840s as a graduate student after learning about Matteuci's work [ 1 10 ] Du Bois Reymond initiated his studies by constructing a very sensitive astatic galvanomet er consisting of about one kilometer and thousands of turns of wire [ 9 10 ] He first used his galvanomete r to confirm Matteuci's finding that current flows between intact and injured muscle surfaces and, in addition, extended the finding to nerve [ 3 ] Using his galvanometer to measure currents through a known resistance he used Ohm's law, which was established in 1827 by Georg Ohm (Germany; 1789 1854), to dete rmine that wounded surfaces always had an electrically negative potential relative to uninjured surfaces [ 7 ] Du Bois Reymond discovered that when electrical stimulation was applied to the positive surface of nerve and muscle, the electrical potential between the injured and uni njured surfaces at that point is reduced, and that the potential of the outer membrane surface actually became negative relative to a distant uninjured surface. He called this reduction in electrical potential "negative variation" and in the mid late 1840 s discovered that the point of reduced potential actually travels along the surface as a wave of "relative negativity", although his instrumentation lacked sufficient time resolution to measure the velocity of the impulse [ 1 7 11 ] Nonetheless, Emil du Bois Reymond was the first to observe and document what would become known as the action potential [ 1 7 10 ] Based on his belief that current flowed between the uninjured surface of a muscle and its tendon during muscle contraction not unlike the flow of current observed between injured and intact muscle surfaces [ 10 ] du Bois Reymond formulated one of the earliest hypothesies in cluding polarization, resting electrical potentials, their discharge during activation, and

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22 "electromotive particles" [ 10 ] as factors underlying the conduction of bioelectric signals [ 7 ] Although many of the specific details of his hypothesis turned out to be incorrect, his ideas were important as they suggested perhaps the first physical mechanism based on experimental observations, explaining what had been referred to simply as "animal spirits" for hundreds of years. Although the nerve impulse was beginning to be understood in physical terms, the velo city of bioelectric signal propagation was widely believed to be immeasurable [ 3 ] Hermann von Helmholtz (Germany; 1821 1894) created an apparatus that was capable of measur ing the duration between stimulation of a point on a nerve and the resulting muscle contraction. In 1849 he compared the latency of mu scle contraction with stimulation at various points along the nerve and determined the impulse velocity in frog nerves to be approximately 27 meters/second, a speed that seemed unexpectedly slow for a purely electrical event [ 5 12 ] This observation led some to doubt the electrical nature of the nerve impulse, with some suggestions that it was fundament ally a chemical event with outward electrical manifestations, while others viewed the result as consistent with du Bois Reymond's hypothesis that nerve conduction of electrical signals involved rearrangements of charged molecules that were more complex tha n simple passive current flow in a wire [ 7 ] Du Bois Reymond passed the challenge of measuring the velocity of the "negative variation" to his student Julius Bernstein (Germany; 1839 1917). In 1868, Bernstein succeeded in not only measuring the velocity of the nerve impulse, b ut also in measuring its time course and in providing its first graphical demonstration using his differential rheotome [ 1 11 ] Importantly, he showed that the propagation velocity of the

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23 "negative variation" observed by du Bois Reymond closely matched the speed of the nerve impulse measured by von Helmholtz, reestablishing confidence in the idea that elect rical activity underlies nerve activity. Bernstein realized that a reduction in electrical potential between injured and intact nerve surfaces was comparable to that of a propagating nerve signal, or that excitability was associated with a decline in the electrical potential driving currents. Based on this fundamental observation, he hypothesized that the membrane is electrically polarized at rest, with the inside negative relative to the outside, and that the action potential is a self propagating loss o f this polarization. In 1902, Julius Bernstein formulated his "membrane theory" in which he was the first to apply Walther Nernst's (Germany; 1864 1941) theory of ion diffusion and electric potentials (1889) to biological membranes, correctly suggesting t hat the origin of membrane polarization was due to a higher concentration of potassium ions inside the cell than outside and a selective resting membrane permeability of potassium. He suggested that a temporary reduction in membrane resistance for all ion s occurred during nerve and muscle excitation, during which time ions flowed down their electrochemical gradients leading to a depolarization of the resting membrane potential [ 7 ] During this time, the composition of the cellular membrane was unknown, but as early as 1899 Charl es Overton (England; 1865 1933) proposed the concept of a lipid membrane based on his observation that lipid soluble dyes penetrate cells more readily than water soluble dyes [ 13 14 ] By 1925, the plasma membrane was proposed to be composed of a lipid bilayer by Evert Gorter (The Netherlands; 1881 1954) and Francois Grendel [ 5 15 ]

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24 Henry Bowditch (USA; 1840 1911), working with Carl Ludwig in Germany, determined that the action potential of frog heart was an all or nothing phenomenon in 1871 since contractile response occurred only after a stimulus above threshold intensity, but remained constant even as stimulus intensity increased [ 7 ] However, this phenomenon did not appear to apply to striated muscle since contraction intensity inc reased proportionately to stimulus intensity regardless of stimulus application through the motor nerve or directly on the muscle. In 1902, Francis Gotch (England; 1853 1913) hypothesized this graded response by striated muscle was due to an increased num ber of excited fibers rather than an increased response amplitude of individual fibers. Shortly after, Keith Lucas (England; 1879 1916) showed that individual fibers of striated muscle follows the same all or nothing phenomenon as cardiac muscle by carefu lly dissecting the frog dorsal cutaneous muscle of the frog to fewer than 20 active fibers and showing that increased stimulation intensities produced contraction in discrete steps [ 7 ] Edgar Adrian (England; 1889 1977), a former student of Keith Lucas, made the first electrical recordings of individual nerve fibers in 1926 after meticulous dissection [ 16 17 ] He showed that a stre tched muscle sends sensory information through the nerve. He observed that when the load on the muscle was increased only the frequency of action potentials increased while the amplitude, duration, and velocity of each pulse remained constant. Adrian's e xperiments provided the first direct evidence of the all or nothing character of the action potential in nerves [ 7 17 ] In the early 1920s, Joseph Erlanger (USA; 1874 1965) and Herbert Gasser (USA; 1888 1963) were the first to feed the output of electrical amplifiers into a cathode ray tube oscilloscope in electrophysiological experiments, allowing for the precise display of high

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25 frequency signals in real time [ 16 18 ] Recording from peripheral nerves in variou s animals, Erlanger and Gasser discovered that the shape of the action potential changed as the recording electrode was moved away from the stimulation electrode in a manner that was dependent on axon diameter, with decreased electrical resistance as diame ters increased. They discovered that action potentials travel at different speeds; signals in relatively thick motor nerves were conducted much faster than signals in thinner pain nerves. In 1937, Alan Hodgkin (England; 1914 1998), attempting to measure the decrease in membrane resistance during the action potential that was predicted by Bernstein's membrane theory, instead made the first measurements showing the passive spread of a local electrical response along the nerve fiber resulting in increased e xcitability [ 12 19 ] This finding was consistent with the local circuit theory proposed by Ludimar Herm ann (Germany, 1838 1914) in the 1870s [ 7 ] Hod g kin's experiments also showed that conduction velocity of the nerve impulse was dependent on the conductivity of the extracellular solution. John Zachary Young (England; 1907 1997) discovered the usefulness of giant squid axons in electrophysiology in 1938 [ 20 ] and just one year later, Kenneth Cole (USA; 1900 1984) and Howard Curtis used squid axons to experimentally confirm the prediction by Bernstein that a decrease in membr ane resistance occurs during the action potential, by making extracellular impedance measurements of a high frequency sine wave applied during the action potential [ 5 21 ] Both Hodgkin and Andrew Huxley (England; 1917 present) and Curtis and Cole independently inserted electrodes inside the giant axon and succeeded in directly measuring the transmembrane pot ential in the

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26 resting state; the interior of the axon was about 50 mV more negative than the electrical potential on the exterior. The existence of the resting membrane polarization hypothesized by Bernstein was confirmed in these studies, however only pa rt of Bernstein's theory was supported. Unexpectedly, the membrane potential overshot the 0 mV level by several tens of mV during excitation, suggesting that a selective membrane permeability, rather than a general permeability decrease, occurred during t he action potential. In fact, Bernstein had observed that the amplitude of the action potential exceeded that of the current measured between the intact and injured surfaces of a nerve. This suggested that the action potential may be more than a destruct ion of the resting electrical condition of the nerve, but Bernstein did not pursue this observation [ 7 11 ] Hodgkin and Bernard Katz (German, but emigrated to England; 1911 2003) showed that the amplitude of the squid action potential decreased in amplitude when external sodium concentrations were reduced, suggesting that a selective permeability of sodium ions is involved in the action potential [ 7 22 ] Bernstein's hypothesis was revised to include a select ive permeability increase of sodium ions during the action potential, which flowed down their electrochemical gradient into the cell to produce membrane depolarization. Although unknown to them at the time, Charles Overton previously reported that externa l sodium ions are essential for the nerve impulse and he hypothesized that excitation required the exchange of sodium and potassium ions in the early 1900s shortly after Bernstein proposed his theory [ 5 23 ] The nature of the action potential precluded study of the relationship between membrane current and membrane voltage since any current large enough to cross threshold also produced changes in

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27 membrane potentials. A method of controlling the membrane voltage was needed and in 1949, Cole and George Marmont developed the voltage clamp. The voltage clamp was used shortly afterwards by Hodgkin and Huxley, in collaboration with Katz, culminating with their famous publication in 1952 that provided final unequivocal evidence that nerve impulses are fundamentally electrical events. The voltage clamp essentially works by very rapidly determining and applying th e feedback current required to keep the transmembrane potential constant during an electrical event; the amount of current injected to maintain the transmembrane potential is of the same magnitude, but opposite polarity of the current actually flowing acro ss the membrane. When Hodgkin and Huxley held membrane potentials at hyperpolarized levels, the recorded currents were inward as expected based on the flow of potassium ions down their electrochemical gradient toward the equilibrium potential. However, w hen membrane potentials were depolarized, above a threshold, the initial phase of the current was still inward and then followed by an outward current. This observation implied that the membrane properties changed upon depolarization allowing an ion to fl ow down a pre existing electrochemical gradient produced by the metabolic activity of the cell. In their experiments, Hodgkin and Huxley altered the external concentrations of sodium and potassium ions allowing them to analyze the membrane current in separ ate components. The initial inward current was found to be carried by sodium ions, and the delayed outward current by potassium ions. They fit a series of differential equations relating the permeabilites of these two ions to membrane potential and time, which were able to account for most of the properties of the action potential, such as the time

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28 course, all or nothing character, refractory period, and the effect of temperature [ 19 23 24 ] Chemical Neurotransmission, Receptors, and Ion Channels First histological identification of neurons with stains oc curred in the mid 1800s by Gabriel Gustav Valentin (Switzerland; 1810 1883), Christian Gottfried Ehrenberg (Germany; 1795 1876), Johannes Purkinje (Czech Republic; 1787 1869) and Albert von Kolliker (Switzerland; 1817 1905). These were followed by descrip tions of the motor endplate by Wilhelm Kuhn (Germany; 1837 1900) in 1862 and descriptions of processes later known as dendrites and axons that extended from neurons by Otto Dieters (Germany; 1834 1863) in 1865. The silver staining method developed by Cami llo Golgi (Italy; 1843 1926) in 1873 greatly enhanced histological and anatomical studies, but did not provide non disputable evidence whether connections between neurons are continuous or discontinuous. Scientists using the same method came to different conclusions, leading to the development of the neuron doctrine by Santiago Ramon y Cajal (Spain; 1852 1934) and the reticular theory by Golgi [ 3 ] In 1856, Claude Bernard showed that skeletal muscle contractions could not be produced by nerve stimulation of muscle treated with curare, and in 1897, Charles S cott Sherrington (England; 1857 1952) coined the term "synapse" to describe the apparent lack of continuity between the sensory neurons and motor neurons in his studies of reflexes and W allerian degeneration [ 3 ] Thomas Elliott (England; 1877 1961), a student of John Langley, is probably the earliest to sug gest that a chemical is released upon sympathetic nerve stimulation. His studies demonstrated that adrenaline mimicked the effect of nerve stimulation at all smooth muscle and glands innervated by sympathetic nerves. He suggested that "adrenaline might b e the chemical stimulant liberated on

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29 each occasion when the impulse arrives at the periphery" [ 25 26 ] In 1914, Henry Dale (England; 1875 1968) identified nicotinic and muscarinic components of autonomic responses induced by acetylcholine ( ACh ) through his studies of sympathetic and parasympathetic nerves, but at the time "had no ev idence at all that ACh was a constituent of any part of the animal body" [ 27 28 ] In 1921, Otto Loewi provided the first demonstration of chemical transmission in the peripheral nervous system, between the vagal nerve and heart [ 17 ] His experiment demonstrated that a substance released by the vagal nerve, which he called "vagusstoff", was capable of slowing the heart rate of an isolated heart. Loewi later identified vagusstoff as ACh in 1926 [ 2 ] In 1929 Dale and co lleagues isolated ACh from ox and horse spleen [ 29 ] and shortly after were able to show that ACh is liberated" from stimulated nerve endings by demonstrating the presence of ACh in acetylcholinesterase treated perfusates of muscle tissue and ganglionic synapses [ 28 30 31 ] Although chemical transmission in the peripheral nervous system was more or less accepted by the 1930s, electrophysiologists questioned the general applicability of these f indings to the central nervous system The debate went on for a number of years and became known as the war between the "Soups and the Sparks". The issue became resolved when intracellular glass microelectrodes (sharp electrodes) and the amplifiers to de al with the high resistances became available, which allowed for the measurement of transmembrane potentials in muscle fibers and neurons. In 1950, Paul Fatt and Katz's intracellular recording at the motor endplate demonstrated that the change in membrane potential upon stimulation was far larger than expected due to circulating currents [ 32 ] John Eccles (Australia; 1903 1997), at one time a lea der of the "Sparks" group, demonstrated that direct

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30 inhibition of the nerve impulse in spinal chord is associated with hyperpolarization of the postsynaptic mem brane, and that the hyperpolarization was not induced by external electrical stimulation in 1952 He considered this as evidence in favor of chemical transmission [ 33 ] Eccles and his group continued with their work, which provided descriptions of the ionic basis of inhibitory and excitatory postsynap tic potentials and demonstrations of ACh release in the CNS [ 34 ] Paul Fatt and Bernard Katz, also in 1952, showed that quantal changes in the electrical potential, called minia ture endplate potentials occurred during voltage recordings of the motor endplate (treated with tetrodotoxin to reduce action potentials) and showed statisti cally that this is described if neurotransmitter is released in quantal events [ 35 36 ] The developmen t of electron microscopy and its application to biological tissue in the mid 1950s finally provided the definitive evidence in favor of neuron theory and also strong support for chemical neurotransmission [ 2 37 ] The images clearly showed an extracellular space between two relatively swollen surfaces of distinct neurons with small vesicles in the pres ynaptic terminal, suggesting that the quantal miniature endplate potentials observed by Fatt and Katz were due to the release of single pre synaptic vesicles filled with neurotransmitter. In 1965, Katz and Miledi demonstrated that voltage gated calcium ch annels open following an action potential invasion of the pre synaptic terminal, and that calcium is necessary for the vesicular release of neurotransmitter [ 38 ] The concept of a receptor was established much earlier from the independent work of Paul Ehrlich (1854 1915) and John Langley (1852 1925) in the late 1800s [ 39 ] Ehrlich, working with selective histological stainin g believed that the staining process relied on the chemical interaction of the dye and a substance on the cell, and later, he

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31 developed side chain theory. He proposed that the cell produces side chain s that bind to toxins and other chemical substances, ba sed on his observations of the immune response [ 39 40 ] Langley, a pharmacologist, studied a competitive int eraction between atropine and pilocarpine on salivary secretion in 1878 [ 41 ] In 1901, he showed that nicotine continued to work on ganglia even after degeneration of the nerve endings and later in 1905 coined the term "receptive substance" in reference to the action of nicotine and curare on muscle tissue [ 42 ] Archibald Vivian Hill (England; 1886 1977), as a student of Langley, studied the relationship between nicotine and curare concentrations and contraction of the frog abdominal muscle and derived the equation that is commonly attributed to Langmuir in 1909 to explain his results [ 43 ] One year later, he refined the equation to include the Hill coefficient, which provided a better fit of his results regarding oxygen binding by hemoglobin in various saline solutions [ 44 ] Alfred Joseph Clark (England; 1885 1941) used Hill's equations in the 1930s to develop receptor theory, which established principles of receptor mediated responses, such as saturation, reversibility, stereoselectivity, ti ssue specificity, and explained the relationship between ligand concentrations versus biological responses in terms of mass action and receptor occupancy by ligand in a quantitative way [ 45 ] Receptor theory was expanded to include competitive antagonists with the work of John Gaddum (England; 1900 1965) and Heinz Schild (England; 1906 1984) in the 1930s through 1950s [ 46 ] [ 47 49 ] This framework was used to classify many pharmacological agents before there was an understanding of how the receptors mediated their response. In 1956, Robert Stephenson proposed his explanation of partial agonism by treating the binding of agonist and the ability to produce a response afterward as

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32 separate steps; he introduced the concepts of affinity and efficacy [ 50 ] One year later del Castillo and Katz proposed the first kinetic mechanism of a receptor mediated response, incorporating an inactive agonist bound state, to explain their observation that decamethonium can be both "depolariz ing and curarizing" [ 51 ] In 1961, Wyman had the initial idea that sub s trates stabilize specific conformations of an enzyme to which they have greatest affinity; the idea o f proteins as allosteric molecules was developed more fully by Monod, Wyman and Changeux in 1965. The allosteric theory was soon applied to the end plate (nicotinic) receptor as early as 1967 and has been used extensively since [ 52 53 ] The del Castillo Katz mechanism and M onod Wyman Changeux model of protein allostery have been usefully applied in the stud y of ligand gated ion channels, but these concepts were proposed at a time when the existence of the ion channel was still a likely suspicion. Although Hod g kin and Huxley definitively demonstrated the electrical nature of the nerve impulse, the mechani sm of ion permeation across the membrane was unproven. In the early 1950s, the increased membrane permeability of ions during excitation was hypothesized to occur either through aqueous pores in the membrane or via ion carrier molecules [ 36 54 ] The identification of tetraethlyammonium in 1957 [ 55 ] and tetrodotoxin in 1964 [ 56 ] which selectively block the sodium and potassium components of the action potential, suggested that separate permeation pathways existed for the two ions. In 1960, Takeuchi and Takeuchi applied the voltage clamp to the motor endplate and demonstrated that the muscle type nicotinic receptor expressed there regulates a non selective permeation pathway for cations [ 57 ] Initial hard evidence for ion channels was obtained from experiments with artificial lipid

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33 membranes. In 1962, Paul Meuller and colleagues reported that the electrical resistance of a lipid bilayer could be reduced by several water soluble proteins [ 58 ] Hladky and Haydon first directly observed ion c hannels in artificial lipid bilayers formed by antibiotics in 1970 [ 59 60 ] Around this time, Bertil Hille (USA; 1940 present) studied the relative permeabilities of over twenty cations with varying physico chemical properties through voltage gated sodium channels. He suggested that sodium channels were aqueous pores and that hydrogen bond interactions betwee n spheres of hydration around individual sodium ions are critical for passage through the "selectivity filter" of the ion channel [ 61 62 ] The first measurements of the currents (mean open times and conductances) through individual ion channels expressed in mammalian cells were performed in the early 1970s by Bernard Katz and Ricardo Miledi through analysis of the noise produced by application of ACh to a voltage clamped motor endplate [ 63 64 ] Katz and Miledi suggeste d that the fluctuations of noise were caused by the "elementary ACh current pulse" and the "ion gate", meaning in today's terms the random opening and closing of single ion channels. They estimated a single channel conductance of 100 pS, a value higher th an expected for a charge carrier and which helped confirm that ion permeation was through an aqueous pore in the membrane. The first direct measurement of single ion channels in animal tissue was made from den ervated frog muscle fibers in 1976 by Erwin Ne her (Germany; 1944 present) and Bert Sakmann (Germany; 1942 present) [ 65 ] Their approach was similar to that which Neher had used previously to record currents from small areas of neuronal cell bodies [ 66 ] They used glass micropipettes with a s mooth tip of 3 5 microns in diameter, which allowed them to reduce background

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34 noise by recording from small patches of membrane. The recording circuitry required modification from typical voltage clamp amplifiers since just one electrode would be used to record the voltage and inject current, while keeping amplifier noise to a minimum. Sakmann and Neher found that upon contacting the surface of muscle fibers (often treated with collagenase and protease) with their micropipette, a seal could be formed with an electrical resistance in the megaOhm range, which provided sufficient electrical isolation of membrane patch to record square shaped pulses of current with unitary amplitudes of less than 5 pA. At last, the current from an individual ion channel had b een observed [ 67 ] The quality of recordings were still limited by noise and patch stability, and a breakthrough was made when it was discovered that very high resistance seals above 10 gigaOhms in resistance could be made by applying li ght negative pressure to the interior of the micropipette after contact with the cell. The robustness of this seal was found to be sufficiently strong to withstand rupture of the membrane under the micropipette, which allowed for recordings from entire ce lls and from excised membrane patches. This allowed for control of the transmembrane potential, and in the case of excised patches concentrations, control of saline solutions on both sides of the membrane [ 68 ] The single channel data collected with this new technique required novel methods for interpr etation, most of which were developed by Alan Hawkes and David Colquhoun (England; 1936 present) Using these methods, Colquhoun and Sakmann provided one of the first single channel kinetic analyses, in which they determined time constants of a plausible kinetic mechanism from recordings of single endplate nicotinic receptors [ 69 70 ] The patch clamp met hod allowed for recording from macroscopic, as well as currents from individual ion channels and very

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35 quickly became one of the preferred electrophysiological methods. The patch clamp method also allows for electrophysiological recordings to be made from individual neurons, contributing much to our knowledge about properties of neurons: the types of receptors they express and their roles in neuronal circuits. Sinc e the discovery of ACh as the first neurotransmitter, many others have been discovered, incl uding norepinephrine, serotonin, dopamine, GABA, and glutamate. Most of these were identified in the nervous system throughout the 1950s, but were shown to be neurotransmitters later. The development of radiolabeled ligands has allowed receptors for most of these transmitters to be localized to specific areas of the nervous system. In addition, peptides, adenosine triphosphate, nitric oxide, and others have been discovered to have neurotransmitter activity [ 2 ] The molecular biology revolution of the 1980s led to the cloning of the receptors for these neurotransmitters, as well as many other ion channels gated by voltage and mechanical stimuli, through meth ods utilizing protein sequencing, homology screens, or functional expression [ 71 72 ] The ligand gated i on channels database currently lists over 550 published sequences of subunits from many species belonging to the Cys loop, glutamatergic, and ATP gated superfamilies (http://www.ebi.ac.uk/compneur srv/LGICdb/LGICdb.php). The first images of an ion channe l were of the nAChR in the late 1980s [ 73 ] In 1998, the first high resolution structure of an ion channe l, the potassium KscA channel was published [ 74 ] Since then several structures of other channels have been solved [ 75 76 ] As a young scientist in the 2000s, it is hard to imagine a time when the nature of the nerve impulse was not considered to be electrical. Even mor e, it is difficult to

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36 envision a time when ion channels were unknown or unproven. It is easy indeed to take for granted the efforts of many scientists and the knowledge that today seems so common sense. Two classes of receptors were identified that resp ond to ACh distinguished by pharmacological profiles first described by Henry Dale. The agonist nicotine activates one of these receptor classes whereas the other class is characterized by the ability to be activated by muscarine and inhibit ed by atropi ne. Unlike nAChRs, muscarinic ACh receptors are not coupled directly to ion channels, and they mediate their response on the relatively slow time scale of milliseconds to seconds through intracellular signals transduced via G Proteins, resulting in severa l possible outcomes which may include the indirect opening of ion channels. I will focus on one of many ion channel families known, the nAChRs with particular emphasis on the 7 nicotinic receptor subtype.

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37 CHAPTER 2 NICOTINIC ACETYLCHOL INE RECEPTORS: INTRODUCTION The Cys Loop Superfamily of Ligand Gated Ion Channels The Cys loop superfamily of ligand gated ion channels includes receptors that are gated by ACh GABA, glyc ine, and serotonin in mammalian cells and all likely evolved from a common ancest or [ 77 ] This superfamily has its name because all the proteins in it have a loop of 13 amino acid residues formed by two disu lfide linked cysteine residues [ 78 ] Although these channels are activated by different agonists and can be permeable to either cations or a nions, they share considerable sequence homology and have similar transmembrane topologies and basic functionality. The finding that inhibitory (anion permeable) and excitatory receptors (cation permeable) are structurally related wa s unexpected [ 79 81 ] All ligand gated ion channels produce biological signals through their ability to bind an agonist, which translates into receptor motion that gates an aque ous ion channel pore and allows charged ions to move across the cell membrane according to pre established electrochemical gradients created by the metabolic activity of a living cell. The functional homology of the Cys loop superfamily was demonstrated t hrough fusion of aspects of the serotonin gated 5HT 3 receptor with the 7 nAChR producing functional chimeras [ 82 ] In addition, the 7 nAChR can be converted t o an anion selective channel by substituting as few as three amino acids in the pore domain with those found at the homologous positions in GABA A or glycine gated receptors [ 83). Due to the fact that more is known about nAChRs than any other ligand gated receptor, they often have been considered as the prototype for studies of other members of the Cys loop superfamily [ 84 ]

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38 nAChRs of muscle and electric organs The muscle type nAChR from the electric organs of eels (Electophorus electricus) and rays (Torpedo californica or marmorata) and the neuromuscular junction became the first and most fully charac terized neurotransmitter receptor, largely due to the abundance and accessibility of the receptor provided by these tissues. The discovery of cobra and bungarus venom toxins with very high binding affinities enabled these receptors to be the first affinit y purified [ 85 86 ] The purification of the electric organ nAChRs in milligram quantities enabled biochemi cal studies that determined the receptor is a pentameric complex consisting of distinct protein subunits. These subunits were called $ and # in order or increasing molecular weight, and found in the stoichiometry of 2 "$# [ 87 88 ] Receptor purification also led to partial N terminal protein sequencing of the subunits [ 89 ] whic h was used to derive nucleic acid probes used to clone the receptor subunits [ 90 92 ] The related subunits of the homologous receptor expressed in mammalian tis sues were subsequently cloned [ 93 ] An additional subunit known as % was shown to replace the $ subunit during the development of skeletal muscle, resulting in new biophysical properties such as increased channel conductances and shorter mean open times [ 94 ] The muscle type nAChR was also the first ligand gat ed ion channel to be imaged using electron diffraction techniques due to the high density of receptor expression in electric organ membranes, which can be treated to form two dimensional crystalline arrays [ 95 96 ] General Features of nAChR Structure Learned from Muscle type nAChRs Early predictions of general subunit structure and orientation in the membrane we re based on hydrophobicity analysis of the primary amino acid sequence. These

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39 predictions were tested by numerous experiments using mutagenic, biochemical, immunological, microscopic, and electrophysiological techniques and have turned out to be reasonabl y accurate [ 92 97 98 ] Images of the elect ric organ nAChR obtained from electron diffraction show that the five subunits of the pentameric complex are arranged in a circular manner to form a central pore The major elements of a nAChR subunit include a large N terminal extracellular domain (which accounts for nearly half of the subunit), followed by three closely spaced transmembrane helices, a large cytoplasmic domain, fourth transmembrane domain and a short extracellular C terminus. Fitting of voltage clamp, ion flux, and binding data with pre dictions from theoretic al receptor activation mechanisms suggested that the muscle type nAChR contained two agonist binding sites [ 99 102 ] Affinity labeling and mutagenesis studies suggested the two agonist binding sites of muscle type nAChR are located in the extracellula r N terminal domain at the # interface and at the $ or % interface [ 103 105 ] Studies using affinity labels identified the presence of two adjacent cysteine residues in the subuni t that contribute to agonist binding sites [ 78 ] These cysteines are now known to be part of a loop known as the C loop, which partially wraps around the outside of the adjacent subunit and constricts over the subunit interface when agonist is bound by the receptor [ 106 ] The agonis t binding site is characterized as a hydrophobic pocket of aromatic amino acids that provide an electrically negative surface which stabilizes the agonist receptor complex through cation & interactions [ 107 ] In addition to the C loop, the subunit contributes the aromatic residues Tyr 93, Trp 149, Tyr 190, and Tyr 198 to form the prim ary surface of the binding site [ 108 ] The non subunit forms the complimentary surface of the binding site, and contains a critical tryptophan res idue at

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40 position 57 in the # subunit and position 55 in the $ and % subunit. The 1 subunit does not contribute amino acids directly to the agonist b inding pocket, but it still influences receptor properties through its contribution to the ion channel pore and by affecting concerted conformational shifts of the pentameric protein [ 78 ] The location of the 1 subunit in the pentamer in relation to the # and $ (or % ) pairs is unproven ; several studies suggest the $ subunit is located between the two subun its [ 109 111 ] but another study suggests the # subunit is located between the two subunits [ 112 ] In addition to the agonist binding sites, the N te rminal domain also contains the characteristic Cys loop, glycosylation sites, and t he main immunogenic region Residues from the top third of the first transmembrane helix and entire second transmembrane helix line the ion channel while the third and fou rth transmembrane domains form a hydrophobic protein core and interact with the cell membrane. The nAChR ion channel pore is permeable to small monovalent and divalent cations, such as sodium and calcium, which create an excitatory or depolarizing signal to the cell when the ion channel is opened. The intracellular cytoplasmic domain between the third and fourth transmembrane helices is by far the least characterized region of the channel, and shows the least amount of conservation between subunit s. Cyto skeleta l attachment points, as well as consensus sequences for serine/threonine and tyrosine phosphorylation sites have been found in this region, which appear to provide a mechanism for modulation of channel function [ 113 117 ] The crystallization of the acetylcholine binding protein (AChBP) a soluble protein important in regulating extracellular ACh concentration in freshwater snails has further confirmed many of the general structural features of nAChR subunits discussed above.

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41 Given that crystallization of membrane associated proteins remains a challenge, the atomic resolution structures of AChBP in apo form and bound to various ligands have been used ext ensively to create homology models of nAChRs [ 118 121 ] The AChBP consists of a homomeric structure with sequence homology to the extracellular N terminal agoni st binding domains of nAChRs, and a lthough the solved structures of AChBP are undeniably valuable, the utility of the AChBP is limited by the facts that the protein lacks transmembrane and intracellular domains of nAChR subunits, and the images are static structures of dynamic proteins. Neuronal nAChR Subunits Oligonucleotide probes designed from muscle type 1 sequence were used to identify the first neuronal nAChR subunit through low stringency screen ing of a PC12 cell cDNA library [ 122 ] Several other homologous AChR subunits were discovered in short order through similar screening methods of rat and c hick cDNA libraries [ 123 ] Although these subunits became known as neuronal nAChR subunits because they were cloned from neuronal like PC12 cells or cDNA libra ries derived from brain tissue [ 124 ] it has become clear that some of t hese subunits are expressed in non neuronal tissue as well, such as microglia, peripheral macrophages, skin, and lungs [ 125 126 ] The neuronal nAChR subunits were classified as if they contained adjacent cysteine residues in the C loop, homologous to those in the muscle type subunit that were shown to be critical elements of agonist binding sites [ 127 ] or subunits if the adjacent cysteines were absent and numbered in the order of discovery. Much of our knowledge of receptor structure, function, and pharmacology has been learned through studies of heterologously expres sed receptors, which was made possibl e with the cloning of the

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42 nAChR subunits. Nine neuronal nAChR subunits ( 2 10) and three subunits ( 2 4) have been cloned to date. The number of possible subunit combinations that can form functional receptors is large, but there appear to be some ge neral rules. The neuronal receptors consist of two general classes, heteromeric receptors or homomeric receptors. Heterologous expression studies have indicated that 2, 3, and 4 subunits require co expression with 2 or 4 for function. Neuronal hete romeric AChRs contain two agonist binding sites formed by the interface of and non subunits. Alpha6 subunits usually assemble with 2 and/or 4, but can also assemble with 3 and other subunits as well. Neuronal 3 and 5 subunits do not appear to form functional agonist binding sites, but rather function as structural or modulatory subunits similar to the 1 subunit of muscle type nAChR [ 128 ] Although 5 subunits contain the vicinal cysteine residues in the C loop, they lack other conserved residues in the N terminal domain that contribute to agonist binding sites. When 5 is assembled into 4 2, 3 2, or 3 4 receptors it can increase agonist sensitiv ity, calcium permeability, and alter decay rates of macroscopic currents [ 129 130 ] An additional source of functional and pharmacological diversity comes from the fact that heteromeric receptors consisting of one type of subunit, and one type of subunit can exist in multiple stoichiometries, such as ( 4) 2 ( 2) 3 and ( 4) 3 ( 2) 2 The ( 4) 2 ( 2) 3 receptors have high sensitivity to agonists as defined by the EC 50 of macr oscopic current responses and relatively low Ca 2+ permeability while the ( 4) 3 ( 2) 2 receptors have relatively low sensitivity to agonist and high in Ca 2+ permeability [ 131 ] The 7, 8, and 9 subunits can assemble into functional homomeric pentamers without subunits. The 8 subunit was cloned in chickens and has not been identified in mammals to date. The homomeric 7 receptor

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43 is one of the most widely expressed nicotinic rec eptor subtypes, whereas 8, 9, and 10 containing receptors are expressed in limited areas such as the retina and ne uroepithilium of the inner ear [ 132 ] In homomeric receptors, each subunit contributes both a primary and complementary face to an agonist binding site so that the total number of agonist binding sites is five [ 133 ] Although 7 9 subunits readily form homomeric nAChRs, these subunits can also form heteropentamers such as 7 2 [ 134 136 ] 7 8 [ 137 138 ] or 9 10 [ 139 ] Another source of potential diversity comes from realizations that partially duplicated 7 gene and splice variants of 7 exist with varying functional properties [ 140 141 ] Physiology, Expr ession, and Functional Roles of Muscle type nAChRs The primary function of the muscle type nAChR at the neuromuscular junction is to depolarize the motor endplate, resulting in activation of a muscle action potential and muscle contraction. In order to pe rform this task effectively, reliable synaptic transmission must occur with the pre synaptic motoneuron. The organization of the neuromuscular junction has evolved to allow the muscle type nAChR to efficiently perform its role. For example, nAChRs are ex pressed in abundance and highly concentrated at the endplate (~20,000 binding sites per square micron) and in close contact with the pre synaptic neurotransmitter release sites. This decreases the effects of diffusio n and allows local ACh concentrations to reach 1 mM in less than one millisecond. In addition, the extracellular matrix of the synaptic cleft has an abundance of acetylcholinesterase. This enzyme rapidly hydrolyzes ACh to produce choline and an acetate group to ensure the signal produced by AC h release occurs only briefly. Each molecule of ACh that is successful in binding the receptor probably only binds once

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44 before being deactivated by acetylcholinesterase. The rapid rise in agonist concentration, the large population of localized nAChRs, a nd brief presentation of a strong stimulus all but ensures the efficient transmission of even very high frequency pre synaptic signals [ 142 ] The abundance of natural toxins that target the muscle type nAChR at the neuromuscular junction, such as bungarotoxin, curare, cobratoxin, and anatoxin a, testifies to the physiological importance of this specialized synapse. Physiology, Expression, and Functional Roles of Neuronal nAChRs General Distinguishing Features of Neuronal nAChRs As early a s the 1940s, pharmacological differences of methonium compounds were observed between nicotinic receptors found at the motor endplate and those found at the autonomic ganglia. For example, ganglionic nAChRs are blocked by hexamethonium and pentamethonium, but not decamethonium whereas nAChRs at the motor endplate are first excited, then desensitized by decamethonium [ 143 145 ] The neuronal nAChRs have higher cal cium permeabilities than muscle type nAChR, with relative sodium : calcium permeability ratios of ~2 to 20 (depending on the neuronal nAChR subtype) and ~0.15, respectively [ 146 149 ] In addition, muscle type receptors conduct current equally well at both hyperpolarized and depolarized potentials (i e have a linear current voltage relationship) while neuronal nAChRs show inward rectification, meaning they conduc t current much better at hyperpolarized potentials than at depolarized potentials. This inward rectification is due to intracellular magnesium ions and positively charged polyamines that block the ion channel at positive potentials [ 150 152 ] Differences in non conserved amino acid residues in or near the channel pore account for the differences between calcium permeabil ity and current rectification Differenc es in subunit composition between individual neuronal nAChR subtypes create

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45 pharmacological diversity allowing for reasonably selective activation, inhibition, or potentiation of specific receptor subtypes. Understanding where the diverse neuronal nAChR s ubtypes are expressed and the functional significance of such diversity continues to be a hot topic of investigation. Peripheral Neuronal nAChRs The nAChRs found in the peripheral nervous system primarily mediate excitatory synaptic transmission at the au tonomic ganglia and the dominant nicotinic receptor subtype invo lved in this important function contains the 3 and 4 subunits. Like heteromeric 4 2 receptors, 3 4 receptors can exist in two stoichiometries and can assemble with 2 or 5 [ 153 ] In many cases the exact stoichiometries of neuronal nAChRs expressed on peripheral ganglia are undefine d. Single ganglionic neurons can express multiple nAChR subtypes. For example, chick ciliary ganglion neurons express 3 containing nACRs in post syn ap tic and peri synaptic locations and homomeric 7 nAChRs in perisynaptic locations only [ 154 155 ] Central Neuronal nAChRs The use of radiolabeled nicotine and bungarotoxin ligands enabled the mapping o f AChR expression in the central nervous system. This approach led to the realization that at least two major populations of neuronal nAChRs exist and that these receptor populations have mostly non overlapping expression patterns; those which bind nicoti ne with high affinity and those which bind bungarotoxin with high affinity [ 156 ] Early studies of heterologously expressed 4 2 receptors suggested they are insensitive to inhibition by bungarotoxin [ 157 ] yet at the same time bungarotoxin sensitive currents in brain tissue were difficult to detect (due to the fact that they desensitize so

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46 rapidly; see below). The neuronal nAChR responsible for bungarotoxin binding was elusive, but was even tually cloned in the early 1990 s with the aid of affinity purification utilizing bungar otoxin and N termin al protein sequencing [ 158 160 ] It has since been established that the majority of high affinity binding sites for nicotine in the brain consist of heteromeric 2 containing receptors, while homomeric 7 receptors account for the majority of high affinity bungarotoxin binding sites [ 154 156 ] Neuronal nAChRs are broadly expressed in many brain regions including the medial habenula, interpeduncular nucleus, retina, lateral and medial geniculate, and cortex. There are at least three major cholinergic subsystems in the brain, and together they innervate nearly every area of the brain. Cholinergic neurons originat ing in midbrain tegmental areas innervate the thalamus and key midbrain dopaminergic centers (substantia nigra pars compacta and ventral tegmental area) and also send descending cholinergic projections into the pons and brainstem. Another cholinergic subs ystem originates in the basal forebrain nuclei (nucleus basalis, diagonal band of broca, medial septal nuclei) and makes widespread cholinergic innervation to the cortex and hippocampus. The remaining cholinergic system arises from a small group of cholin ergic interneurons in the striatum. Together, these cholinergic interneurons make up only approximately 2% of all striatal neurons, but they provide very rich and localized cholinergic signals to specific regions in the striatum and olfactory tubercle [ 161 ] In sharp contrast to cholinergic signaling at the motor endplate and at autonomic ganglia, the majority of cholinergic release sites in the central nervous system rele ase ACh diffusely through volume transmission [ 162 163 ] rather than at focused synaptic sites. One striking example of an area where cholinergic synaptic transmission was expected

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47 to be found, but was not is a fiber tract running from the medial habenula to interpeduncular nucleus known as the fasciculus retroflexus of Meynert. Despite the fact that this pathway contains a high density of cholinergic fibers and high expression of choline acetyltransferase, excitatory synaptic transmission in this area is glutamatergic, not cholinergic [ 164 166 ] The release of ACh likely occurs in relatively low frequency (4 10 Hz) theta rhythms and the diffusion of ACh is largely limited by acetylcholinesterase. This enzyme is common in the central nervous system, but doe s not always match up with ACh release sites [ 167 ] The precise temporal and spatial dynamics of ACh concentrations are still unknown in the brain, and efforts continue to understand this important parameter. Some estimat es suggest that steady state extracellular ACh concentrations in the brain are in the low nM range, but it is very likely that ACh concentrations vary significantly from one microdomain to another [ 168 ] At any rate, the presentation of ACh in the brain appears to be vastly different from that at the neuromuscular junction and autonomic ganglia. There are only a few reports of nAChR mediated fast synaptic trans mission, and in many of these cases the evidence is equivocal [ 166 169 171 ] The primary mediators of fast synaptic transmission in the central nervous system are glutamatergic and GABAergic systems. The widespread distribution of nAChRs and observations that they are expressed primarily at somatic, pre terminal, pre synaptic, peri synaptic, and extra synaptic sites, rather than at post synaptic sites in the brain is consistent with the observation of di ffuse cholinergic release sites [ 156 172 175 ] It has become widely accepted that nicotinic receptors expressed in the brain play roles in modulation of other neurotransmitter

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48 systems through enhancement of neurotransmitter release, modification of neuronal ex citabililty, and influences on synaptic plasticity. Numerous studies with isolated synaptosomes and electrophysiological studies in brain slices have shown that neuronal nAChRs modulate the release of glutamate, dopamine, noradrenaline, and serotonin from brain regions including the medial habenula, interpeduncular nucleus, thalamic nuclei, midbrain limbic re gions, cortex, and hippocampus [ 176 179 ] If the a pplication of nicotinic ligands increase the frequency of spontaneous currents in a tetrodotoxin resistant, calcium dependent, and nAChR antagonist sensitive manner, the results are generally interpreted to be m ediated by pre synaptic nAChRs [ 180 181 ] By taking advantage of the unusually large size of the presynaptic terminals at the developing chick ciliary ganglion, inward currents evoked by nicotine, block ed by bungarotoxin, and resistant to tetrodotoxin were recorded di rectly from the nerve terminal [ 178 ] Neuronal nAChRs influence electrical activity in nearly every area of the brain where they participate in aspects of neuronal signaling involved in attention, learning, memory, and development [ 182 184 ] Disruption of nicotinic signa ling mechanisms contributes to Alzheimer's disease, Parkinson's disease, s chizophrenia, dementia with Lewy bodies, forms of epilepsy, pain, anxiety, attention deficit disorders autism, and addiction [ 185 187 ] Research is ongoing to determine functional roles of individual nAChR subtypes and their influence in specific brain regions for these diverse indications. Initial interest in neuronal nAChRs as thera peutic targets came from early observations that nicotine enhances attention and memory performance, combined with observations that a selective loss of cholinergic neurons occurs in Alzheimer's disease. At the behavioral level, the effects of nicotine an d other nAChR agonists on cognition

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49 have turned out to be varied. Overall, the data suggest that nAChRs are involved in select forms of memory, such as working memory and episodic memory, and appear to be particularly important when tasks are difficult or when subjects are impaired [ 188 189 ] High Affinity Binding Sites The most widely expressed neuronal nA ChR subunit is 2, which accounts for the vast majority (>90%) of high affinity binding sites for nicotine in rodent brain [ 190 ] This finding was confirmed when mice lacking the 2 subunit lost most, but not all, high affinity nicotine binding [ 191 192 ] The 2 subunit most commonly associates with the 4 subunit in rodent brain, but can assemble with m ost of th e other nAChR subunits as well [ 193 ] Th e 2 subunit is more widely expressed in the primate brain than it is in rodent brain, and 2 2 receptors may constitute a major nAChR subtype in primate and human brain [ 194 ] Labeling of 2 containing receptors with the monoclonal antibody mAb 270 was highest in the interpeduncular nucleus, thalamic nuclei, superior colliculus, and medial habenula, with more moderate labeling in the presubiculum, cerebral cortex, substantia nigra (pars compacta), and ventral tegmental area [ 156 ] Alpha3 containing receptors are prevalent in autonomic ganglia, but also have limited expression in brain regions such as the medial habenula, pineal gland, spinal cord, and retina [ 132 ] The habenula is a region of diverse an d concentrated nAChR expression [ 195 ] The mechanism of nicotine addiction is unknown, but strong evidence suggests that nicotine targets the dopaminergic mesolimbic and nigrostriatal pathways, where it modulates the firing modes, frequencies, and release of dopamine by midbrain

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50 dopaminergic neurons [ 196 198 ] The rewarding influence of nicotine on these pathways is supported by the finding that nicotine administration is reduced when dopamine release is blocked in the nucleus accumbens [ 199 ] Beta2 containing nAChRs with 4 and/or 6 are expressed abundantly in these areas directly on dopaminergic, and also on GABAergic neurons. The net result of nicot ine on dopaminergic neurons appears to be increased excitation and decreased inhibition [ 200 ] Desen sitization of the heteromeric receptors on inhibitory GABAergic neurons becomes absorbing with a prolonged low level nicotine stimulus, thus reducing an excitatory drive of the inhibitory neuron to result in less inhibition of the dopaminergic neuron. At the same time, the low affinity 7 nAChRs on glutamatergic terminals enhance glutamate releas e onto the dopaminergic neuron [ 175 187 201 ] Although heteromeric nAChRs initially open with high probability [ 202 ] they accumulate into absorbing high affinity desensitized states that resist further activation more readily at low agonist concentrations than 7 nAC hRs [ 203 205 ] It is well established that chronic nicotine upregulates the expression of 2 containing nAChRs [ 206 ] A surprising finding from 2 or 4 knockout mice is that they survive much better than predicted. Beta2 knock out mice fail to self administer nicotine and show nicotine enhanced avoidance learning behavior [ 191 ] In addition, aged mice show impaired learning in the Morris water task, but appear otherwise grossly normal. It is still not clear whether the diversity of nAChR subtypes represents redundancy or a specific functional design. In contrast, knockout of 3 is lethal; likely due to the key role it plays in the autonomic ganglia of the peripheral nervous system.

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51 Low Affinity Binding Sites: 7 nAChRs In autoradiographic binding experiments receptors are exposed to ligand for prolonged time periods and accu mulate in desensitized states. If the desensitized state shows low affinity for the ligand, the receptors will lack high affinity binding for agonist and a signal will fail to be produced. This is the case for the homomeric 7 nAChR. In contrast, these receptors bind the antagonist bungarotoxin almost irre versibly. Labeling of 7 nAChR with radiolabeled bungarotoxin and in situ hybridization show high expression in brain regions recognized in cognitive function, including hippocampus (CA1, CA3, dent ate gyrus), and cortex (especially layer V), as well as subcortical limbic regions (ventral tegmental area and substantia nigra), hypothalamus, and thalamic nuclei [ 132 156 207 ] In addition, 7 nAChR expression occurs in reticular thalamic nuclei in macaques and i n human brain tissue [ 208 210 ] The reticular thalamic nuclei are important in mediati ng attention and sensory gating, processes wh ich are negatively affected in s chizophrenia. In the hippocampus, the 7 nAChR is expressed particularly highly on GABAergic interneurons, where it modulates inhibitory/disinhibitory tone onto glutamatergic neu rons [ 211 ] and to a lesser extent is express ed on glutamatergic terminals. Distinguishing Functional C haracteristics of the homomeric 7 nAChR The 7 receptor, one of highest expressed nicotinic receptor subtypes in brain and also expressed in non neuronal cells, is commonly thought to be the ancestor of the other nicotini c genes [ 212 ] One of the most distinguishing functional characteristics of the 7 nAChR is the strong dependence of response ki netics on agonist concentration [ 213 214 ] At relatively high a gonist concentrations peak 7 mediated currents

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52 increase with agonist, but the net charge (area under the response curve) remains relatively unchanged. This is indicative of increased synchronous activity, but not increased overall receptor activation by high agonist concentrations This phenomenon is clearly demonstrated through comparison of the concentration response relationships of peak currents versus net charge and is accounted for by a form of desensitization that is readily and rapidly entered in a concentration dependant manner [ 213 ] Unlike heteromeric nAChRs, the binding sites of 7 appear to cooperate in a negative fashion to produce less channel activation under conditions of high fractional occupancy of the agonist binding sites. The 7 ion channel is characterized by an extremely low probability of being open (P open ) during an agonist application. Records of currents mediated by individual 7 receptors show that openings occur rarely, and when they do occur are isolated events usual ly lasting 100 seconds or less [ 215 216 ] A precise measurement of 7 P open has not been possible due to the inability to know the number of functional receptors in a population that contribute to a res ponse, but the open conformation of the 7 receptor appears to be an energetically unfavorable and unstable condition of the protein molecule. Over time, lower agonist concentrations may produce low level sustained currents that produce more net charge than a response evoked by higher agonist concentrations that quickly favors desensitization. Recovery from desensitization occurs rapidly upon the removal of agonist and receptors readily return to an activatible resting state, consistent with the observ ation that 7 receptors lack high affinity binding for traditional nicotinic agonists. Desensitization has provided a real challenge in the study of 7 nAChR in native tissues, often making it impossible and at best very difficult to detect contributions of 7 to pr ocesses under

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53 study [ 217 ] The ability of 7 nAChR to be activated selectively by choline is another characteristic that sets it apart from other nAChRs and may have important physiological significance. In hypothalamic t uberomammilary neurons, spontaneous firing activity can be modulated by bath applied choline [ 218 ] Choline, an essential nutrient, is a key component of phospholipids found in the plasma membrane and also a product of acetylcholinesterase activity. Although choline concentrations are typically well below the EC 50 for activation at 10 M, this low concentration may pro vide a tonic signal to 7 nAChRs, potentially producing low level activation or pre desensitization. Under conditions of stroke or injury choline concentrations in the brain can increase up to 100 M [ 219 ] Neuronal nAChRs are known for their relatively high calcium permeability, and this is especially true for the 7 nAChR. The relative permeability of calcium ions is generally reported to be in the range of 10 20 times greater than the permeability of sodium ion [ 160 177 220 ] The calcium permeability of the 7 nAChR is often considered comparable to that of the NMDA type glutamate receptor; this may be of critica l importance since 7 nAChRs pass current at resting transmembrane potentials while NMDA type receptors do not due to the extracellular magnesium block The high permeability of nAChRs to calcium may allow relatively low numbers of nAChRs to exert signifi cant effects in cellular compartments of small volume, such as in pre synaptic terminals. The hi gh calcium permeability of 7 has been shown to arise from specific residues on either side of the pore, which can be mutated to eliminate calcium permeability [ 221 ] In addition to directl y mediating calcium influx, 7 nAChRs have been shown to induce secondary changes in intracellular calcium through calcium

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54 induced calcium release from internal stores through ryanodine and/or IP 3 receptors [ 222 226 ] The high calcium permeability has brought a lot of attention to the 7 nAChR, particularly since it has implications for intracellular signaling, gene expression, neurotransmitter release, synap tic plasticity, and cytotoxicity. The calcium signal appears to promote either survival or death, depending on the amplitude and duration of the signal [ 225 227 228 ] Relatively weak stimulation was shown to protect differentiated PC12 cells from nerve growth factor and serum deprivation in a manner that depe nded on protein kinase C and phospolipase C, but high agonist concentra tions produced toxicity [ 223 227 ] The t oxicity was attenuated by pre application, but not post application, of methyllycaconitine ( MLA ) whereas protection was only blocked by a 10 minute post application of MLA. This observation suggests that toxicity is produced through rapid activation of t he ion channel, whereas neuroprotection occurs through slower mechanisms, probably involving activation of i ntracellular signaling pathways [ 227 229 ] Excitotoxicity produced by excess calcium entry is well known to promote to neuronal death and degeneration [ 230 ] Implications of 7 nAChR in Pathophysiology Nicotine positively modulates a number of brain functions linked to cognition, such as attention, arousal, learning, and memory functions [ 182 184 ] The importance of the neuronal cholinergic function to learning and memory was first recognized when muscarinic ACh receptor antagonists were found to impair memory in rats [ 231 ] and in human young adults [ 232 ] Subsequently, studies of brains from patients with advanced age or Alzheimer's disease consistently found abnormalities in basal forebrain cholinergic neurons that correlated with the level of cognitive decline. Consequently,

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55 the hypothesis that loss of cholinergic function in the central nervous system significantly contributes to the cognitive decline associated with age and AD was developed [ 233 ] Although so me debate exists regarding the roles of the cholinergic system in the early stages of Alzheimer's disease [ 234 238 ] degeneration of cholinergic neurons in Alzhe imer's disease is firmly established. The cholinergic hypothesis of cognitive decline is supported by a considerable amount of evidence which includes: (a) reports that both muscarinic and nicotinic receptor antagonists impair memory performance in a vari ety of behavioral paradigms in rodents, non human primates, and humans [ 234 ] (b) lesions in animals that damage cholinergic input to the neocortex or hippocampus from the basal forebrain structures diminish performanc e in the same memory tasks affected by cholinergic blockade [ 239 ] (c) ACh synthesis and release is reduced in aged animals [ 240 243 ] (d) aged and Alzheimer's patients show high sensitivity to the memory impairing effects of cholinergic antagonists [ 244 2 46 ] and (e) measurements of cholinergic function with in vivo imaging methods reveal multiple aspects of the cholinergic system are compromised in Alzheimer's and memory impaired patients [ 247 250 ] The observation that cholinergic neurons are lost in Alzheimer's disease led to the development of acetycholinesterase inhibitors as a therapy. To this day, acetylcholinesterase inhibitors and memantine (a weak non competitive blocker of NMDA type glutamate channels) are the only therapies for Alzheimer's disease approved by the Federal Drug Administration. At best these treatments work temporarily to alleviate symptoms without altering the progression of the disea se.

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56 Normal aging, traumatic brain injury, Parkinson's disease, and schizophrenia often have a component of significant cognitive decline [ 251 256 ] and the rapies aimed at restoring, maintaining, and/or protecting the cholinergic system through nicotinic ligands appear promising and remain under pursuit [ 257 ] While it is true that degeneration of cholinergic pathways is not solely responsible for neurodegenerative disease and that both muscarinic and nicotinic mediated signaling are affected by such degradation, the 7 nicotine receptor is singled out as a therapeutic target due to the substantial body of data suggesting a) that 7 is a primary mediator of nicotine induced neuroprotection [ 258 266 ] b) 7 selective agonists exert a cyto protective influence against trophic factor deprivation [ 227 265 267 268 ] ethanol toxicity [ 269 270 ] glutamate excitotoxicity [ 271 ] amyloid beta(1 42) toxicity [ 272 ] and hypoxia [ 273 274 ] on neurons and cultured cells [ 223 227 270 275 281 ] (c) 7 selective agonists enhance cognitive function [ 188 255 257 282 296 ] an d (d) 7 nAChRs do not appear to mediate addiction [ 297 298 ] A number of repor ts have indicated that th e 7 nAChR interacts with amyloid beta (1 42), the primary component of senile plaques found in the brains of Alzheimer's patients. The data have not always been reproducible or clear, likely due to difficulties in reconstituting active amyloid beta, but do impl icate a connection between 7 and the disorder. In addition to cognitive deficits, 7 nAChRs have been implicated in s chizophrenia through sensory gati ng deficits. Individuals with s chizophrenia are significantly more likely to smoke tobacco than non schizophrenics [ 299 300 ] ; this observation has been generally seen as self medication and has sti mulated interest in nAChRs as a therapeutic target in the disease. Schizophrenics often have impaired auditory gating,

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57 as demonstr ated by transcranial recordings [ 301 ] In normal individuals, auditory stimuli presented in quick success ion are processed such that the response from the second stimulus is greatly attenuated relative to the first. In schizophrenics, there is much less suppression of the response to the second stimulus, which is indicative of failed auditory processing [ 302 303 ] The implication of this deficit is that hallucinations, delusions, and paranoia may result from i ntegration of unfiltered stimuli by the brain. Genetic linkage studies have mapped sensory gating deficiencies to the chromosomal locus 15q13 q14, which contains the 7 gene [ 304 306 ] and may be due to diminishe d promoter efficacy of the 7 gene [ 307 ] In addition, 7 nAChR expression is often reduced in the hipp ocampus and thalamus of postmortem brains [ 305 308 ] Consistent with these observations, a number of 7 agonists and positive allosteric modulators ( PAMs ) have been shown to improve auditory gating deficits in preclinical models [ 299 ] and in humans [ 303 ] Therapeutic Targeting of the 7 nAChR through Positive Allosteric Modulation The term "allosteric" was first introduced following observations that bacterial enzym es were inhibited by the end product of synthetic pathways, even though the end product had limited structural similarities with the enzyme active site substrate [ 309 ] The inhibition appeared to be non competitive with substrate, which led to hypotheses that the non competitive inhibitor produced conformational alteration s in the protein [ 310 ] and the formulation of the well kn own Monod Wyman Changeux model of prote in allostery [ 311 313 ] The basic concept proposed that proteins are dynamic structures existing in multiple discrete functional states or conformations, all of which are accessible to the protein under resting conditions. Binding of a ligand alters the resting

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58 equilibrium by reversibly stabilizing the protein in the conformation to which the ligand has greatest affinity. The conformation of each subunit was pr oposed to be constrained by the other subunits, and protein symmetry always conserved. Therefore, the binding of a ligand at one site was predicted to alter the affinity of the other binding sites within the oligomer for the ligands. The model was soon a pplied to hemoglobin to describe the cooperative nature of oxygen binding, and to proteins involved in signal transduction, including membrane receptors as diverse as G protein coupled receptors and ligand gated ion channels including nAChRs [ 312 313 ] During the last 20 years, intensive effort has been put forth to identify agonists that selectively a ctivate the 7 nAChR. Numerous compounds have been discovered and characterized [ 314 ] Recently an alternative avenue of targeting the 7 nAChR for therapeutic purposes has been appreciated with the discovery of PAMs This approach may offer the means to overcome some of the factors that limit the efficacy of agonists, such as the low P open and rapid desensitization, potentially with sig nificant benefit for processes that depend on channel activation [ 291 ] In addition, PAMs may syner gize and augment natural neurotransmitter mediated signals rather than oppose or attempt to replace them since a PAM requires the presence of an agonist to function. Further, the binding sites for PAMs are located away from well conserved agonist binding domains, which may enhance the identification of compounds with desirable subtype selectivity. The sedative hypnotic drugs, such as the barbiturates and benzodiazepines, which target GABA A receptors, provide a proven example of allosteric modulator based therapy, with ion channels from the same superfamily as nAChRs [ 315 ]

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59 Positive allosteric modulators selective for 7 share some of the putative therapeutic activities ascr ibed to 7 agonists, including enhancement of memory related behavioral performance in the eight arm radial maze, social recognition, and Morris water maze tasks [ 316 317 ] and normalization of sensory gating deficits [ 316 318 321 ] The intrinsically low P open of 7 makes it a good candidate for positive allosteric potentiation. However, since 7 is a receptor with high calcium permeability, it must also be considered whether extreme increases in 7 channel activation may lead to unexpected, and potentially undesi rable, effects. The identified 7 PAMs have considerable diversity, ranging f rom proteins to small molecules [ 322 ] Even the small molecule PAMs vary significantly in structure, as well as in properties and probably mechanisms of the modulation [ 323 324 ] Gronlien et al ., 2007 proposed that 7 PAMs be divided into two classes, type I and type II, based on the functional properties of modulation. Following the rapid application of agonists, all PAMs appear to increase receptor sensitivity to agonists, current magnitudes, and empirical Hill coefficients; the type I PAMs (for example 5 HI, NS 1738, or CCMI) do so with little or no effect on the basic onset and decay kinetics, or shape, of the response, while the type II PAMs (for example PNU 120596, TQS, or A 867744) markedly slow response decay kinetics and can even activate receptors that have been desensitized by applications of high agonist concentrations or by application of agonists like GTS 21 that produce residual inhibition or desensitization [ 316 318 325 328 ] Since the two classes of 7 modulators were prop osed, additional PAMs have been identified that display properties intermediate to the type I and type II classes [ 320 321 329 ]

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60 Putative allosteric modulator binding sites of GABA A receptors have been identified in the N terminal extracellular domains at subunit interfaces not form ing orthosteric agonist binding sites (benzodiazepine binding site) and in the transmembrane region (barbiturate, alcohol, neurosteroid, general anesthetic binding sites [ 330 334 ] ) This information, along with the differing functional profiles of type I and type II PAMs, may be seen as consistent with the hypothesis that multiple allosteric sites also exist on 7 The site where calcium binds to potentiate res ponses on 7 was localized to the N terminal extracellular domain [ 335 336 ] In addition, the crystal str ucture of galantamine bound to AChBP [ 337 ] mutagenesis studies [ 3 38 ] and computer docking simulations [ 339 340 ] provide further evidence for nAChR allosteric sites in t he extracellular domain, some of which may be related to benzodiazepine binding sites on GABA A receptors [ 337 341 ] Using 7 /5 HT 3 chimeras, the N terminal extracellular domain of 7 has been shown to be sufficient for potentiation by the type I PAMs NS 1738 [ 342 ] and 5 HI [ 343 ] but not the type II PAM PNU 120596 [ 342 ] In addition, PNU 120596, but not NS 1738, potentiated currents (ACh evoked) in a reverse 5 HT 3 / 7 chimera c ontaining 7 TM regions [ 342 ] These studies also provide evidence th at the extracellular 7 M2 M3 loop is im plicated in potentiation by geni stein and NS 1738 [ 342 343 ] Other studies usi ng 7 /5 HT 3 chimeras suggest that TM1 TM3 regions are critical for potentiation by PNU 120596 and the type I modulators LY 2087101 and ivermectin [ 344 345 ] Furthermore, mutations at several 7 amino acid residues, which are hypothesized to contribute to an intrasubunit cavity within the four transmembrane domains, significantly reduced potentiation by PNU 1 20596, LY 2087101 [ 344 ] and

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61 ivermectin [ 345 ] Through site directed mutagenesis, these studies together identify potentially important differences in modulator interactions with the receptors. For example, the A225D m utation reduced potentiation by PNU 120596 significantly more than it reduced potentiation by LY 2087101 [ 344 ] and the S276V mutation had no effect on potentiati on by PNU 120596 [ 344 ] but conferred inhibitory properties to ivermectin [ 345 ] Based on their work with 7 /5 HT 3 chimeras, mutagenesis studies, blind computer docking simulations, and evidence that an intrasubunit tra nsmembrane site appears to be important for potentiation of glycine and GABA receptors [ 331 346 349 ] Young et al ., 2008 proposed the intrasubunit cavity is a highly conserved modulatory site of Cys loop ion channels [ 344 ] a hypothesis which has been supported by oth ers [ 331 350 ] The fact that CCMI was discovered from a library of GABA A PAMs (that bind away from the benzodiazepine site ) provides further support for the existence of conserved allosteric sites and/or mechanisms [ 316 ] The findings that the extracellular domain a ppears to be sufficient for potentiation by some type I PAMs (NS 1738 and 5 HI), but not others (ivermectin and LY 2087101) suggest that more than one mechanism may produce the type I potentiation profile, and perhaps the same applies to the type II profil e. Positive allosteric modulators have been demonstrated to potentiate many types of 7 mediated responses that may be important within biological systems. For example, ERK1/2 phosphorylation was enhanced in PC12 cells by A 867744 [ 351 ] and PNU 120596 [ 351 353 ] with several structurally diverse agonists. Several studies have provided evidence that activation of ERK1/2, the prototypical mitogen activa ted kinase,

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62 is important for 7 mediated protection from death in PC12 cells [ 223 352 355 ] and have implicated ERK1/2 in cognitive funct ions [ 355 359 ] Several PAMs, including 5 HI, PNU 120596, SB 206553, and A 867744, enhance agonist evoked 7 responses from hippocampal CA1 stratum radiatum i nterneurons [ 318 320 326 360 361 ] and glial cells [ 362 ] in acute brain slices. Spontaneous and choline or ACh induced increases in GABAergic inhibitory post synaptic currents were enhanced by 5 HI [ 363 364 ] PNU 120596 [ 318 ] LY 2087101 [ 365 ] and A 867744 [ 326 ] in hippocampal neurons. JNJ 1930942 increased the amplitude of excitatory post synaptic potentials in hippocampal dentate gyrus [ 321 ] In cerebellar slices, 5 HI enhanced ACh induced frequency increases of excitatory post synaptic currents mediated by glutamate [ 366 ] PNU 120596 (10 M) potentiated 7 induced increases in [ 3 H] D aspartate release from prefrontal cortex synaptosomes, as well as [ 3 H] dopamine release from prefrontal cortex in vitro and in vivo [ 367 ] The nicotinic facilitation of long term potentiation was enhanced by PNU 120596 [ 368 ] and JNJ 1930942 [ 321 ] in rat dentate gyrus. Physiological concentrations of choline (~10 M) and 1 5 M PNU 120596 activated 7 containing receptors in tuberomammillary neurons and hippocampal CA1 pyramidal neurons sufficiently to depolarize a cell and facilitate the firing of action potentials [ 217 369 ] Supporting the potential therapeutic significance of these in vitro assays, NS 1738 [ 317 ] and CCMI [ 316 ] have been shown to enhance performance in behavioral measures of cognitive function; and CCMI [ 316 ] PNU 120596 [ 318 319 ] SB 206553 [ 320 ] A 867744 [ 319 ] and JNJ 1930942 [ 321 ] have been shown to reverse auditory gating deficits in drug induced or DBA/2 models with systemic administration to rodents,

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63 indicating that these PAMs have sufficient pharmacokinetic propertie s to modulate brain 7 in vivo and that a sufficient level of endogenous agonist is present in relevant brain regions. Pharmacokinetic studies generally suggest that brain concentrations of PAMs are low compared to the EC 50 for potentiation determined in vitro [ 316 317 319 321 ] However, since 7 has an intrinsically low P open even modest potentiation of 7 mediated signals may be sufficient to produce significant in vivo effects. A recent study has provided evidence that simultaneous modulation of 5 GABA A and 7 nicotinic receptors may funct ion to synergistically enhance long term potentiation in rodent brain slices and memory related behavior in vivo [ 370 ] This study provides proof of concept evidence that molecules engineered to modulate multiple targets might provide an optimized approach for specific therapeutic purposes. Activation of 7 can have either protective or toxic effects depending on the mode of stimulation [ 227 ] Positive allosteric modulators have been identified which increase the open probability of 7 by several orders of magnitude, inviting the question of whether PAMs may take the activation of this receptor subtype, which has high permeability to the natural catalytic ion calcium, to dangerously high levels. Calcium mediated toxicity has been rep orted in SH SY5Y cells and mice expressing mutant forms of 7 that display dramatically prolonged responses after stimulation [ 371 373 ] In vivo toxicity profil es for PAMs are lacking, but there are some in vitro experiments that suggest type I and type II PAMs may have different profiles. Ng et al ., 2007 showed that 24 hour exposure to the type I PAM CCMI did not reduce the viability of SH SY5Y cells, whereas t he type II PAM PNU 120596 did under the same conditions and in a MLA sensitive manner [ 316 ] Prior to the toxicity assays the authors tested the

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64 cells to verify the presence of functional receptors and successful modulation by PNU 120596 and CCMI. A similar exper iment with stable 7 expressing GH4C1 cells showed that the type II PAM PNU 120596 was toxic, while JNJ 1930942 was not toxic in the same paradigm [ 321 ] Howev er, Hu et al 2009 failed to detect toxic effects in undifferentiated PC12 cells and cortical neurons after treatment with the type I PAM CCMI, but they also did not detect any toxic effects with the type II PAMs PNU 120596 or A 867744 [ 374 ] The reason for the contradictory results is unclear, but two shortcomings of this study are that functional 7 expression and potentiation were indirectly evaluated, and only one agonist, PNU 282987, was tested at only one concentration, 10 M.

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65 CHAPTER 3 METHODS cDNA Clones Human nAChR receptor clones were obtained from Dr. Jon Lindstrom (University of Pen nsylvania, P hiladelphia PA). The human ric 3 clone was obtained from Dr. Millet Treinin (Hebrew University, Jerusalem Israel) and co expres sed with 7 to improve the levels and speed of receptor expression [ 375 ] Mouse muscle nAChR 1, 1, $ and # clones used for receptor expression in Xenopus oocytes were obtained from Dr. Jim Boulter (UCLA, Los Angeles, CA), and the mouse % clone was provided by Dr. Paul Gard ner (University of Massachusetts Medical School, Worcester, MA). The mouse muscle cDNA clones in pRBG 4 used for transfection of BOSC 23 cells were obtained from Dr. Steven Sine (Mayo Clinic, Rochester, MN). The red fluorescent protein clone, pDsRed N1, wa s obtained from Clontech (Palo Alto, CA) and used to identify successfully transfected BOSC23 cells. Chemicals Solvents and reagents were purchased from Sigma Aldrich Chemical Company (St. Louis, MO), and Fisher scientific (Pittsburg, PA). 4OH GTS 21 was obtained from Taiho (Tokyo, Japan). AR R17779 was provided by Critical Therapeutics, Inc. (Lexington, MA). MTSEA was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). PNU 120596 was synthesized by Dr. Jingyi Wang as described in [ 376 ] [ 125 I] bungarotoxin was kindly provided by Dr. Ralph Loring (Northeastern University, Boston, MA). Unlabeled bungarotoxin was purchased from Biotoxins, Inc. (Saint Cloud, FL). Cell culture supplies were purchased from Life Technologies (Grand Island, NY) Fresh ACh and MTSEA stock solutions were made each day of

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66 experimentation. MTSEA stock solutions were made in water, kept on ice, and diluted just before experiments. PNU 120596 stock solutions were prepared in DMSO, stored at 20 C, and used for 30 day s. PNU 120596 solutions were prepared fresh each day at the desired concentration from the stock in saline solution. Site Directed Mutations Mutations of selected amino acids were introduced using the QuikChange kit from Stratagene (La Jolla CA) accord ing to the manufacturer's instructions. Sequences were confirmed with automated fluorescent sequencing at the University of Florida core facility. Preparation of RNA for Injections into Xenopus laevis Oocytes Subsequent to linearization and purification o f cloned cDNAs, cRNA transcripts were prepared in vitro using the appropriate mMessage mMachine kit from Ambion Inc. (Austin, TX). Expression in Xenopus laevis Oocytes Mature (>9 cm) female Xenopus laevis African frogs (Nasco, Ft. Atkinson, WI) were used as the source of oocytes. Frogs were maintained in the Animal Care Services facility of the University of Florida, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and the University of Florida Insti tutional Animal Care and Use Committee approved all procedures Prior to surgery, frogs were anesthetized by placing the animal in a 1.5 g/L solution of 3 aminobenzoic acid ethyl ester (Sigma, St. Louis, MO) for 30 minutes. Oocytes were removed from an a bdominal incision. In order to remove the follicular cell layer, harvested oocy tes were treated with 1.25 mg/mL collagenase (Worthington Biochemical Corporation, Freehold, NJ) for 2 hours at room temperature in calcium free Barth's

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67 solution (88 mM NaCl, 1 mM KCl, 2.38 mM NaHCO 3 0.82 mM MgSO 4 15 mM HEPES (pH 7.6), 12 g/L tetracycline). Subsequently, stage 5 oocytes were isolated and injected with 50 nl (5 20 ng) each of the appropriate subunit cRNAs. Wild type and mutant 7 receptors were routinely co injected with the cDNA for human ric 3, an accessory protein that improves and accelerates 7 expression [ 377 ] without affecting t he pharmacological properties of the receptors. RNA for 4 2 receptor s ubunits were injected at an : ratio of 1:1. Note that the injection of 4 and 2 subunits into the oocytes at a ratio of 1:1 results in a mixture of ( 4) 2 ( 2) 3 and ( 4) 3 ( 2) 2 stoich iometries [ 378 ] Muscle type receptor cRNAs were injected in the ratio of 2 : 1 : 1 # : 1 $ or 1 % Recordings were made 1 to 10 days after injection. Two Ele ctrode Voltage Clamp Electrophysiology Experiments were conducted using OpusXpress 6000A (Molecular Devices, Union City, CA) [ 379 ] OpusXpress is an integrated system that provides automated impalement and voltage clamp of up to eight oocytes in parallel. Oocytes were perfused with Ringer's solution (115mM NaCl, 2.5mM KCl, 1.8mM CaCl 2 10mM HEPES, 1 M atropine, pH 7.2), and agonist solutions were delivered from a 96 well plate via disposable tips, which eliminated any possibility of cross contamination. Drug applications alternated between ACh controls and experimental applications. Perfusion flow rates were set at 2 mL /min for experiments with 7 receptors and 4 mL /min for other subtypes. Unless otherwise indicated, drug applications were 12 s in duration followed by 181 s washout periods with 7 receptors and 6 s with 241 s washout periods for ot her subtypes. Oocytes were voltage clamped at a holding potential of 60

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68 mV. Data were collected at 50 Hz and filtered at 20 Hz. Both the voltage and current electrodes were filled with 3 M KCl. Responses of non 7 wild type and mutant receptors are r eported as peak currents, and responses of 7 wi ld type and mutant receptors are calculated as net charge [ 214 ] Each oocyte received two initial control applications of ACh, and thereafter was rechallenged with ACh at the control concentration following each experimental drug application. For experiments with wild type 7 and mutant 7 receptors, the control ACh concentration was 300 M; except in the case of 7W55Y the ACh control was 30 M because this mutant could not be activated repeatedly by 300 M ACh without rundown. For experiments with non 7 subtypes control applications of ACh were 30 M; except in the case of 4 W 149 2 mutants control ACh applications were 100 M A Ch. The peak amplitude and the net charge [ 214 ] of experimental responses were calculated relative to the preceding ACh control responses in order to normalize the data, compensating for the varying levels of channel expression among the oocytes. For the experiments in chapter 3, the standard MTSEA treatment in the oocyte experiments was 2 mM applied for 60 s, a treatment that appears to produce a maximal effect on receptors expressed in Xenopus oocytes [ 380 ] Mea ns and standard errors were calculated from the normalized responses of at least four oocytes for each experimental concentration. For concentration response relationships, data were plotted using Kaleidagraph 3.0.2 (Abelbeck Software; Reading, PA), and curves were generated as the best fit of the average values to the Hill equation Response = (I max [agonist] n )/([agonist] n +(EC 50 ) n ) where I max denotes the

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69 maximal response for a particular agonist/subunit combination and n represents the Hill coefficient. I max n, and the EC 50 were all unconstrained for the fitting procedures except in the case of the ACh concentration response curves. Because ACh is the reference full agonist, the data were normalized to the obser ved ACh maximum, and the I max of the curve fits were constrained to equal 1. Error estimates of the EC 50 values are the standard errors of the parameters based on the Levenberg Marquardt algorithm used for the generation of the fits [ 381 ] Although some 4 2 concentration response curves were not ideally fit by the single site Hill equation, presumably because 4 2 receptors expressed from RNA injected at a 4: 2 ratio of 1:1 resulted in 4 2 receptors of mixed stoichiometry [ 378 ] in most cases the single site Hill equation provided better concentration response curve fits than the double site Hill equation, and so for consistency, single site fits were gener ated for all 4 2 data sets. Transient Transfection of BOSC23 Cells BOSC 23 cells obtained from American Type Culture Collection (Manassas, VA) were cultured at 37¡C, 5% CO 2 in DMEM supplemented with 10% fetal bovine serum ( FBS ) in the absence of antibiotics. Cells wer e not used for more than 35 passages. One day prior to transfection, cells were plated o nto 12 mm glass coverslips (Fis her Scientifi c, Pittsburgh, PA) coated with poly D l ysine (Sigma, St. Louis, MO). Cells were transiently transfected using Fugene 6 (Ro che, Indianapolis, IN) according to the manufacturer's instructions. In the transient transfection of the mouse fetal muscle type receptors, 1 g of mouse fetal muscle type receptor cDNA ( 2 : 1 :1 $ : 1 # ) in pRBG4 with 0.8 g of the cDNA encoding red fluorescent protein in pDsRed (Clontech, Palo Alto, CA) were added to each 35 mm dish containing cells and coverslips. In the transient

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70 transfection of the human 7 nAChR, 1 g, 0.3 g, and 0.8 g of plasm ids containing cDNA clones of h 7, hric 3, and DsRed, respectively, were added to each 35 mm dish containing cells and coverslips. Experiments were performed 48 to 72 hours after transfection. The red fluorescent protein was used as a marker to identify successfully transfected cells. Generation of HEK293 Cells Stably Expressing H uman 7 and H uman r ic3 Low passage number HEK293 cells were obtained from ATCC ( Manassas, VA ) In order to create a HEK293 s tably expressing the h 7 nAChR, two rounds of stable transfection were performed. First, cell lines were transfected with either h r ic3 or h 7 and several (>20 ) clones that were resistant to 0.15 mg/mL hygromycin (resistance conferred by pcDNA3.1/r ic3 vec tor) or 0.5 mg/mL G418 (resi stance conferred by pCiNeo/ 7 vector) were isolated using cloning cylinders (Sigma St Louis, MO ) and expanded. The transfections were performed with Fugene HD (Roche Indianapolis, IN ) according to manufacturer's directions. The d ay before transfection, 125,000 cells were plated in 35 mm dishes. One g of circular pcDNA3.1 plasmid contai ning the hr ic3 gene or 2 g of circular pCiNeo p lasmid containing the human 7 gene were used in transfection. Following the selective perio d, the hygromycin resistant cell lines and G418 resistant cell lines were maintained in normal growth media supplemented wi th 0.15 mg/mL hygromycin and 0.5 mg/mL G418, respectively. Total RNA was extracted using the SV Total RNA Isolation System (Promega Madison, WI) and the expression of h r ic3 or 7 mRNA was determined by RT PCR The upper and lower primers for h 7, respectively, were TGGACGTGGATGAGAAGAA and TTCCCACTAGGTCCCATTC resulting in an expected product size of 414 base pairs

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71 The upper and lower primers for hric3 respectively, were CCG ATTTCCACCTATGATG and GGCTGCTTCTGTCTCCTTC, resulting in an expected product size of 346 base pairs. The upper and lower primers for GAPDH, respectively, were ACGGATTTGGTCGTATTGG and TGGCATGGACTGTGGTCAT, resulting in an expected product size of 516 base pai rs. N one of the antibiotic resista nt and PCR positive clones for r ic3 or 7 responded to co applications of ACh and PNU 120596 in patch clamp electrophysiology experiments. However, responses from these cells were observed when stably expressing 7 cells wer e transiently transfected with r ic3 on ly, or vice versa, when stable r ic3 cell lines were transiently transfected with 7 alone (not shown). The process of transfection, selection, cloning, and expansion wa s repeated using a stable HEK hr ic3 expressing cell lin e as the starting point. The hr ic3 expressing cell line was tra nsfected with 2 g of circular pCiNeo/h 7 pl asmid. The cells were selected using 0.45 mg/mL G418 and 0.015 mg/mL hygromycin, and thereafter maintained in the same concentration of selective antibiotics. Ten colonies were eventually cloned that were resis tant to both hygromycin and G418 and wer e also positive for both human ric3 and human 7 mRNA by RT PCR. These cell lines were subsequently screened for functional 7 channel expression through whole cell patc h clamp electrophysiology. The clone (clone 1 0) that showed highest functional channel expression, and was easiest to patch clamp, was selected for use in all of the following studies. This cell line will be refer red to as HEK h 7/hr ic3 For normal passaging, c ells were dissociated with a trypsin f ree solution containing 0.02% EDTA in calcium and magnesium free Hank's balanced saline solution ( HBSS ) to avoid non selective damage to the 7 nAChRs expressed on the cell surface. For electrophysiology

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72 experiments, cells were plated on poly D lysine co ated cover slips and were used 1 5 days after plating for experiments. Immunoprecipitation and Western Blot Three days prior to preparing the sample for western blotting, 50,000 cells were plated in a 12 well plate treated with poly D lysine. Cells were washed with ice cold 1% PBS, lysed (lysis buffer, protease inhibitor and phosphatase inhibitor), scraped off the 12 well plate, and transferred into 1.5 mL tubes. The c ells were then sonicated on i ce and incubated overnight at 4¡C with primary antibody ab848, courtesy of Dr. Cecilia Gotti (University of Milan, Italy). The antigen antibody co mplexes were incubated with 50L of pre washed protein A magnetic beads (Millipore Billerica, MA ). After washing, immunoprecipi t at ed protein underwent denaturing elu tion with sample buffer followed by heat ing to 80¡C for 10 minutes. 40 L were loaded into each well and underwent SDS polyacrylamide gel electrophoresis using 10% polyacrylamide, usin g 1L of MagicMarkXP (Life Technologies, Grand Island, NY ). Protein was transferred overnight at 4¡C onto a PVDF membrane. Transfer was confirmed by staining the PVDF membrane with Ponceau S (Bio Rad Hercules, CA ) and the gel with coomassie blue. Ponceau S was removed from the membrane using TBS T. Membrane was blocked at r oom temperature with 5% bovine serum albumin ( BSA ) Overnight incubation at 4¡C of the primary antibody ab849 courtesy of Dr. Cecilia Gotti (University of Milan, Italy) was followed by washing the membrane in TBS T, and incubating it with secondary antibo dy (Abcam Cambridge, MA ) for one hour at room temperature. After washing again with TBS T, Super Signal (Thermo Fisher Scientific Waltham, MA ) was a dded to visualize the protein using the ChemiXRS+ imaging system (Bio Rad Hercules, CA ).

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73 Fluorescence Mi croscopy Untransfected HEK293, HEK h 7/hric3, HEK h 7, and HEK hr ic3 cells were plated on square glass coverslips (Fisher Scientific Waltham, MA ) coated with poly D lysine and incubated at 37%/5% CO 2 in DMEM media with 10% FBS containing the appropriate selective antibiotic for 24 hours pri or to imaging. Cells were treated with 1 g/mL (~125 nM) of Alexa488 bungarotoxin (Life Technologies Grand Island, NY ) for 45 minutes at 37C/5% CO 2 For a control, the HEK h 7/hric3 was also pre incubated with 1 mM nicotine, and then 1 g/mL Alexa488 bungarotoxin was co applied with 1 mM nicotine. After the incubation, the cells were carefully rinsed 4 times PBS to remove any excess Alexa488 bungarotoxin. The cells were then fixed with 4% (v/v) formaldehyde in PBS for 15 minutes, and washed 3 mor e times with PBS before being mounted on coverslips for imaging using VectaShield (Vector Laboratories, Burlingame, CA) mounting media containing the n uclear stain DAPI The slides were imaged immediately using an Olympus DSU IX81 spinning disc confocal m icroscope. The images were obtained using a Hamamatsu C4742 80 12AG Monochrome CCD Camera. Equilibrium Radioligand Binding Assay Two to three days prior to binding assays cells were plated in poly D lysine treated 24 well dishes. Experiments were perfor med when the cells were ~60 80% confluen t The growth media was removed and cells were washed one time with Dulbecco's phosphate buffered saline (DPBS; Life Technologies Grand Island, NY ). [ 125 I] bungarotoxin containing solutions (0.05 nM 7 nM) were a dded to the cells and incubated at room temperature for 3 hours. Non specific binding was determined with

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74 the addition of 1 M unlabeled bungarotoxin in separate wells After the 3 hour incubation, the radioligand was removed and the cells were washed three times with cold DPBS. The cells were then solubilized with 0.1 M NaOH/ 0.1 % SDS and samples were counted in a gamma counter (Beckman Coulter, Brea, CA) Saturation binding curves were fit to the equation, B max [ligand]/(EC 50 + [ligand]), with Kaleida graph 3.0.2 (Abelbeck Software; Reading, PA). Each condition was tested in triplicate in each experiment, and each experiment was repeated three independent times. Cytotoxicity Experiments The cells were maintained in normal growth medium, and experiment s were complet ed in HBSS (Life Technologies Grand Island, NY). One day prior to performing th e toxicity studies, two sets of HEK h 7/hric3 cells from the same passage together with two sets of untransfected HEK293 cells from the same passage were plated in 96 well plates at a density of 15,000 cells per well in normal growth medium grown at 37 ¡ C/5% CO 2 Experimental solutions were prepared in HBSS and applied to the cells. One set of HEK h 7/hric3 cells and untransfected HEK293 cells were placed in an incubator set to 28 ¡ C / 5% CO 2 and the other set of HEK h 7/hric3 cells and untransfected HEK293 cells were placed in an incubator set to 37 ¡ C/ 5% CO 2 Incubations with experi mental treatments were 2 hours since maximal toxicity occurred within 2 hours, as d etermined from separate experiments evaluating the onset of toxicity at various time points during 24 hours following treatments. Following the 2 hour treatment period, the experimental solutions were replaced with 100 L HBSS and 20 L per well of CellTi ter96 solution (Promega, Madison, WI) and incubated for 2 4 hours at 37C with 5% CO 2 after which absorbance readings were made with a microplate

PAGE 75

75 spectrophotomer at 490 nm (BioTek Winooski, VT ). E ach condition in an experiment was tested in triplicate and experiments were repeated on at least 3 independent occasions. Background absorbance was measured from cell free wells and subtracted from all control and experimental test conditions. Absorbance readings from experimental test conditions were normalize d to the absorbance values of untreated / DMSO vehicle controls, which were defined as 100% cell viability. The DMSO was used to dissolve PNU 120596, which is insoluble in water. The highest concentration of DMSO applied with PNU 120596 was limited to 0.3% and this occurred in the 30 M PNU 120596 condi tion. The 20% DMSO and 30 M thapsigargin conditions were used as positive controls. Cells with fewer than 30 passages since transfection were used in all toxicity experiments. Outside out Patch Clamp Electrophysiology Single chan nel currents were recorded in the outside out patch configuration using an Axopatch 200A amplifier (Molecular Devices, Union City, CA) at room temperature. Cells were bathed in an external solution containing (in mM): NaCl ( 165), KCl (5), CaCl 2 ( 2), glucos e (10), HEPES ( 5), atropine (0.001), pH adjusted to 7.3 with NaOH. Patch pipettes (Sutter Instruments, Novato, CA) were pulled to a tip diameter of 1 2 m, fire polished to 5 10 M # coated with SigmaCote (Sigma, St. Louis, MO), and filled with an internal solution containing (in mM): CsCl (147), MgCl 2 (2), CaCl 2 (1), EGTA (10), HEPES (10), Mg ATP (2), pH adjusted to 7.3 with CsOH. No correction was made for the liquid junction potential, which was calculated to be 4.6 mV. Recordings were low pass filtered to 10 kHz with the built in amplifier filter (4 pole Bessel) and digitized at 100 kHz with a DigiData 1440 (Molecular Devices Union City, CA ) using Cl ampex 10

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76 data acquisition software (Molecular Devices Uniion City, CA ). Multiple recordings for each experimental condition were obtained from several transfection and recording dates. Rapid drug application to outside out patches was performed in a sim ilar manner as described by [ 382 ] and [ 383 ] Theta glass (Sutter Instruments Novato, CA ) was pulled, scored, and then broken by hand to create an application p ipette with a diameter of ~120 M (septum thickness: ~ 10 M). The application pipette was mounted to a Burleigh pi ezoelectric stepper (EXFO, Ontario, Canada). The signal sent by Clampex 10 (Molecular Devices Union City, CA ) to the piezoelectric stepper was conditioned by an RC circuit ( = 2 ms) to reduce oscillations and avoid damage to the crystal [ 384 ] Two solution reservoirs (60 mL Monoject syringes; Sherwood Medical, St. Louis, MO) were connected to each channel of the theta glass application pipette via polyethylene tubing. Flow rates from each reservoir and channel of the drug application pipette were equivalent. Solution exchange times were typically 0.4 0.7 ms (10% 90% rise times) and were routinely determined by movement of diluted external solution over an open recording pipette. To maintain undisturbed laminar flow from the application pipette and minimize solution mixing, external saline solution was continuously perfused through the recording chamber (Warner Instruments, Hamden, CT) and the application pipette was positioned such that streams flowing from it would directly enter the aspiration port of the chamber. In addition, the tip of the application pipette was kept free of dirt and/or cell debris by periodic cleaning in a hydrochloric acid solution. All solutions were de gassed under vacuum and passed through a 0.2 m filter to reduce the probability of particles/air bubbles obstructing solution flow and/or damaging the

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77 outside out patch. In the experiments with MTSEA and the MTSEA sensitive mutants (Chapter 5) the chamber was completely emptied and thoroughly rinsed anytime MTSEA entered the chamber, whether the patch survi ved the entire protocol or not, and the coverslip of cells was replaced. Patches were not treated with MTSEA for the experiments in which 10 nM ACh was applied to 1 1 $# L121C mutant receptors. In the case of the experiments with the muscle type nAChR (Chapter 5 ), all patch clamp recordings were processed, idealized, and analyzed with Clampfit 10 (Molecular Devices Union City, CA ). Prior to any analysis, each recor ding was additionally low pass filtered to 5 kHz with a software filter simulating an 8 pole Bessel filter, corrected for baseline drift, and any recorded artifacts or spurious noise were removed. The 5 kHz filter frequency was selected as a compromise be tween reliable event detection and total bandwi dth. A resolution limit of 1.3 x filter rise time was set at 86 sec and imposed on all recordings [ 385 ] Absolute P open (NP open ) values were used as the primary measure of response to ACh for the outside out patch clamp experi ments, in a manner analogous to the net charge measurements made from responses by receptors expressed in Xenopus oocytes. The NP open value was computed for an entire response to ACh, including the non stationary phase of activation by, where I is the recorded current relative to baseline, t is time, i is the mean single channel amplitude, and D is the duration of the ACh application No attempt was made to estimate P open for an individual channel since the total number of activatible channels in a pat ch could not be known with any degree of certainty, and because each patch

PAGE 78

78 served as its own control. Therefore, no kinetic information relating to a single channel is intended by the macroscopic NP open measurement. Single channel recordings were idealize d with half amplitude idealization When simultaneous channel openings occurred, segments of data containing single channel activity were selected so that non co nducting flanking regions were $ 50 ms Apparent subconductances occurred occasionally, but were ignored since they were not obvious in all traces and since they appeared to occur independently of MTSEA treatment. Data from at least 4 individual patches f rom each condition were pooled together to obtain sufficient numbers of events for analysis. Burst analysis was conducted with the intention of defining groups of one or more apparent channel openings that arise from an individual channel. Apparent chann el openings separated by a closed interval less th an the defined t crit of 3.4 ms were called a burst of openings. The t crit value was ca lculated based on the equation, which misclassifies equal proportions of short and long intervals, from fit time cons tants of the closed duration histogram of non MTSEA treated 1 1 $# L121C receptors [ 386 387 ] The t cr it value determined for wild type 1 1 $# receptors was 3.1 ms; small variations in t crit values did not lead to significant changes in burst durations and for consistency the t crit valu e of 3.4 ms applied to the wild type recordings. The value of t crit fo r 1 1 $# L121C patches that received 10 nM ACh was defined as 3.8 ms by the same method. In the case of experiments performed with the human 7 nAChR (Chapter 6), patch clamp recordings were analyzed with Clampfit 10 (Molecular Devices, Union City, CA) and QuB (University at Buffalo, Buffalo, NY) software. Sections of data traces with minimal simultaneous openings and flanked by $ 100 ms of closed time were selected

PAGE 79

79 for analysis. Following initial periods of multi channel openings, single channel bursts po tentiated by PNU 120596 occurred in very obvious groups of openings that were separated by long silent periods. This characteristic eliminated the ambiguity that is normally associated with defining a burst delimiter value or t crit used in burst analysis of single channel recordings [ 387 ] The t crit value used to determine average burst duration intervals was 150 ms; large variations in this valu e had minimal impact on the outcome of burst analysis. To determine intraburst closure, subconductance, and open durations, a total of 40 protracted bursts were selected at random for careful idealization. Data traces were idealized within QuB using the segmental k means method [ 388 ] and were idea lized at a bandwidth of 10 kHz, but the sampling rate was reduced from 100 kHz to 30 kHz to improve the quality of idealization since segmental k mean idealization is generally optimized at or near the Nyquist frequency (F. Sachs, personal communication vi a QuB user forum). Following the automated idealization, the fit was manually inspected event by event and corrections were made as necessary to the idealization. A temporal re solution limit of 1.3 x filter rise time was set at 40 sec and imposed in all analyses. Whole c ell Patch Clamp Electrophysiology Whole cell recordings were recorded using an Axopatch 200 amplifier (Molecular Devices, Union City, CA) In the experiments that involved temperature adjustments, the temperature was controlled with a Warner TC 324B temperature controller (Warner Instruments, Hamden, CT). Cells were bathed in an external solution containing (in mM): NaCl ( 165), KCl (5), CaCl 2 ( 2), glucose (10), HEPES ( 5), atropine (0.001), pH adjusted to 7.3 with NaOH. Patch pipettes (Sutter Instruments, Novato, CA) wer e

PAGE 80

80 pulled to a tip diameter of ~ 2 m, fire polished to approximately 5 M # and filled with an internal solution containing (in mM): CsCl (147), MgCl 2 (2), CaCl 2 (1), EGTA (10), HEPES (10), Mg ATP (5 ), pH adjusted to 7.3 with CsOH. Cells were held at 70 mV. Recordings were low pas s filte red to 5 kHz and digitized at 50 kHz with a DigiData 1440 or DigiData 1322A (Molecular Devices Union City, CA ) using Clampex data acquisition software (Molecular Devices Union City, CA ). A 10 ms test pulse of 10 mV was used to determine access and inpu t resistances prior to each response For experiments performed at room temperature, whole cell recordings were analyzed if access resistances were < 40 megaOhms and membrane resistances were >200 megaOhms. On average, the access resistance, membrane res istance, and cell capacitance values were 15.8 0.6 megaOhm s 1.55 0.15 gigaOhm s and 55.2 6.6 pF respectively. For experiments evaluating the temperature dependence of PNU 120596, these basic criteria were necessarily relaxed at 37 ¡ C. Sweeps with access resistance < 40 megaOhms input resistance > 100 megaOhms holding current < 700 pA were included in the analysis. No attempt was made to compensate for series resistance or for the liquid junction potential although the liquid junction potential wa s calculated to be 4.7 mV. For the ACh concentration response and MLA inhibition curves in Figure 7 4 drug applications were made using a Burleigh piezoelectric stepper (EXFO, Ontario, Canada) in a similar manner as described above with the outside ou t patches, but with a drug application pipette with a diameter of ~350 m. Solution exchange times were 2.6 0.8 ms as determined by measuring holding current shifts (10 90% rise times) upon moving diluted external solution over an open recording pipette each day of experimentation

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81 The open recording pipette was positione d just above the surface of the recording chamber and in a similar arrangement relative to the drug application pipette that occurred during a whole cell recording. It should be stated that the exchange time estimate measured in this manner is probably on ly a lower limit (i.e. fastest time possible) of the solution exchange time achieved to a whole cell during a recording. Three applications of 300 M ACh were appl ied initially followed by 3 test applications of 1 M 3 mM ACh with 60 second interstimulus intervals. The responses from the initial 3 responses to 300 M ACh were averaged, as were the 3 responses from the test concentration of ACh T he averaged response of each cell to the test ACh concentration was normalized to the averaged response to 300 M ACh to compensate for varying levels of receptor expression among individual cells The normalized responses were subsequently adjusted to r eflect the response relative to the maximal normalized ACh evoked response which was defined as 1 as described above with the oocyte experiments Both peak current and net charge responses were measured; net charge responses were measured as the area un der the activation curve during one second of ACh application. Means and standard errors were calc ulated from the responses of 4 8 cells at each test concentration. T he concentration response relationship was fit to the Hill equation using Kaleidagraph 3 .0.2 (Adelbeck Soft ware Reading, PA ) as described above. Experiments for the MLA inhibition curve were performed in a similar manner as the ACh concentration response curve with the initial concentration of ACh being 170 M and test concentrations consi sting of 170 M co application with 3 nM 100 M MLA. No pre applications of MLA were made. Each data point represents the mea n and standard error of 4 6 c ells at each test

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82 concentration and the conc entration response relationship was fit to the Hill eq uation using negative Hill slopes. In all other whole cell electrophysiology experiments, l ocal applications of drug were made using single barrel glass pipettes attached to a picospritzer (General Valve, Fairfield, NJ) with Teflon tubing (11.5 14.5 psi ). The application pipette was placed within 10 15 m of the cell body Drug applications were 3 seconds in duration and were made every 60 seconds. Drugs applied with this method were determined to be diluted 1.5 fold by the time they reached the cell surface (Williams et al ., in preparation). In the temperature experiment s, 3 baseline responses were recorded at room temperature (23.5 ¡ C ) after which responses were recorded as the temperature was increased to 37 ¡ C. Three responses were recorded at 37 ¡ C and then the temperature was returned to 23.5 ¡ C C ells with responses that failed to recover to 50% of the baseline responses upon temperature reduction from 37 ¡ C to 23.5 ¡ C were excluded from analysis; 15 of 70 total cells failed to meet this criterion. Responses were measured as peak currents. In mos t cases, the currents recorded at 37 ¡ C wer e normalized to the initial baseline responses recorded at 23.5 ¡ C In comparing the effect of BSA on potentiation by PNU 120596 at 37 ¡ C responses are shown as both normalized currents and the absolute magnitude o f evoked currents. To test for the statistical significance of BSA on PNU 120596 potentiation at 37 ¡ C the sweeps obtained from each cell at 37 ¡ C were averaged to obtain one mean response from each cell at 37 ¡ C. The mean responses from cells recorded at 37 ¡ C either in the absence or presence of 30 M BSA were then compared with a two tailed student's t test using Microsoft Excel (Microsoft, Redmond, WA)

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83 CHAPTER 4 DIFFERENTIAL REGULAT ION OF RECEPTOR ACTI VATION AND AGONIST SELECTIVITY BY HIGHL Y CONS ERVED TRYPTOPHANS IN THE NICOTINIC ACETYLCHOLINE RECEPT OR BINDING SITE Introduction Sequence analysis of receptor subunits within the Cys loop superfamily, all the way through to prokaryotic ligand gated ion channels, illustrate s remarkable conservation at select sites and implicate great functional significance to aromatic r esidues localized in the ligand binding domain (LBD) of receptors within the Cys loop superfamily [ 389 ] In nAChRs, W 55 and W149 have been identified amongst other aromatic ring containing residues as highly conserved throughout evolution and as contribut ors to th e formation of a hydrophobic agonist binding pocket [ 84 118 390 396 ] Several studies have shown that mutation of highly conserved aromatic residues typically results in decreased efficacy and/or potency for ACh and related ammonium compounds [ 397 400 ] However, observations by Horenstein et al 2007 suggest that mutation of conserved aromatic residues may not result in loss of receptor activity for all ligands, and conserved aromatic residues may differentially regulate receptor activation by select agonists. Specifically, the activation of human 7 nAChR by 4OH GTS 21 an 7 selective agonist, is unaffec ted by mutation of Y188 to phenylalanine while ACh potency is drastically reduced in the mutant 7 receptor The effect of the homologous mutation was qualitatively different in heteromeric 4 2 receptors. While ACh potency was unaffected by the 4Y190F mutation, the efficacy of 4OH GTS 21 relative to Ach was increased at least 200 fold in 4 Y190F 2 receptors compared to wild type [ 401 ] These findings suggest the assumption that all conserved residues play comparable roles in all receptor subtypes may be invalid.

PAGE 84

84 The nAChR LBD is localized at the interface of two subunits; W55 and W149 (human 7 numbering) are found on opposing sides of this interface. In heteromeric receptors such as 4 2 and muscle type receptors, W57 is found on the non subunit (complementary face) while W154 is found on the subunit (primary face) contributing to the bin ding site. As might be expected, the only subunits in which 7W55 and 2W57 are not conserved are subunits that do not form the complementary face, while the only subunits in which 7W149 and 4W154 are not conserved are subunits that do not contribute to the primary face of an agonist binding site (Figure 4 1A). These tryptophans are both found in subunits which can form homomeric receptors ( 7 10 ) since these subunits contribute to both the primary and complementary faces of binding sites in the homomeric receptor [ 402 ] The crystal structure of AChBP isolated from Lymnaea stagnalis which is homologous to the extracellular domain of a homomeric receptor [ 118 ] suggests the indole ring of the W at position 149 (human 7 numbering) is positioned vertically, deeper and slightly higher in the binding pocket relative to W55, which is positioned horizontally at the mouth of the aromatic pocket (Figure 4 1B). In this study site directed mutagenesis and heterologous expressi on in Xenopus oocytes are used to investigate the functional significance of 4W154, 2W57, 7W55, and 7W149 for the activation of homomeric and heteromeric neuronal nAChR by ACh and the structurally diverse 7 selective agonists choline, 4OH GTS 21, and AR R17779 The data suggest that nAChR are likely activated in different manners by structurally distinct agonists and provides insight regarding subtype selective activation of nAChR In particular, some amino acid residues are critical for activation, like 7

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85 W149 (and homologue in other nAChR subtypes), regardless of agonist while others may be involved in regulating subtype selectivity, like 7 W55 and 7 Y188. Results Before the results of this chapter are presented I want to make clear that my contribution to this work was to analyze, interpret, and present the current findings in context of previously published literature. Most of the experiments in this chapter were originally conceived by Dr. Papke and most of the data were collected by OpusXpress techn icians of the Papke Laboratory. Mutation of W55 or W57 of 7 and 4 2 Receptors, respectively, Alters the Pharmacology and Regulates the S electivity of 4OH GTS 21 Two general types of effects resulting from mutations in the nAChR LBD were anticipated. First, that there would be changes in the ability of ACh to promote channel activation; and second, that there would be differences in the relative efficacy of 7 selective agonists compared to ACh. Changes in relative efficacy were of particular interest since the primary goal was to test the hypothesis that 7 s elective agonists may promote activation through mechanisms that are distinct from the activation mechanism invoked by ACh. While measurements of relative efficacy are readily obtainable from macroscopic currents as long as a reliable reference by which t o measure efficacy is employed, effects on the absolute ability of ACh to promote activation are relatively difficult to measure from macroscopic currents since decreases in ACh evoked responses may result from either decreased ACh activity or decreased re ceptor expression /assembly An obstacle in this study for the application of traditional measurements of receptor expression, such as radioligand binding assays, is that mutations of highly conserved LBD residues are arguably equally likely to affect liga nd

PAGE 86

86 binding as receptor activation, potentially rendering results of binding experiments uninterpretable. For example, the 7L119C mutation, which is described in chapter 5, disrupts the binding the bungarotoxin. Absolute Efficacy of ACh With this limitation in mind, the effects of the mutations tested in this study were estimated relative to the absolute effectiveness of ACh to promote channel activation. The comparison was made based on the maximum ACh induced currents from the mutant receptors to the maxim um ACh induced currents in wild type receptors that were injected the same day from the same harvest of oocytes with RNA of comparable amount and quality, as confirmed on denaturing gels. These measurements of maximal ACh induced currents then formed the basis from which efficacies of choline, 4OH GTS 21, and AR R17779, re lative to ACh responses in wild type and mutant receptors, were subsequently determined. Choline and AR R17779 are considered full agonists of 7, activating 7 as efficaciously as ACh, while 4OH GTS 21 is a 7 selective partial agonist. None of these drugs produce significant currents in 4 2 recept ors (Figure 4 2). As seen in Figure 4 3A, calculated ACh maximum responses for 7 W55A and 7W55 to glycine mutants were approximately 2.5 times larger than for wild type, the 7W55 to valine mutant maximum was approximately equal to wild type, and 7W55Y and 7W55F had ACh maximum net charge responses approxim ately one half as large as wild type 7. Homologous mutations were made in 2. Wild type and 2 mutant subunits were co expressed (1:1) with 4. As with the 7W55 mutants, the 4 2 W57A, G, V, Y a nd F

PAGE 87

87 mutants gave functional responses to ACh, and 4 2 W57 arginine, serine, and threonine mutants did not (Figure 4 3B and D). However, none of the 4 2 mutant peak cur rents were as large as the wild type responses. The calculated maximum ACh responses of the functional mutants were on average appr oximately one tenth of the wild type calculated maximum ACh peak response (Figure 4 3C). Nonetheless, the ACh potencies were greater for some of the 4 2 W57 mutants than for the wild type (see Figure 4 5). R elative Efficacy of 7 S elective Agonists Compared with ACh The mutation of 7W55 to the non aromatic amino acids A, G, and V resulted in reduced potencies for all the agonists tested (Figure 4 4A C, Table 4 1). Although mutation of 7W55 to G produced a decrease in the potency of ACh, choline, and AR R17779 (22 fold, 14 fold, and >92 fold increases in EC 50 values, respectively, Table 4 1), maximal responses to these agonist were larger than those of wild type receptors. Maximal ACh responses of 7W55G mu tants were about 2.5 fold higher tha n maximal ACh responses in wild type receptors (Figure 4 3A). Relative to the maxim um ACh induced currents in wild type and mutant receptors choline efficacy was increased in 7W55G mutants (Figure 4 4B). A saturating I max for choline relative to ACh was not possible to achieve due to the low potency of choline for the W55G mutant. In contrast to the results seen with ACh and choline, 7W55G receptors did not produce measurable responses to 4OH GTS 21 in the concentrati on range tested. Mutation of 7 W55 to the other non aromatic residues, A and V, likewise resulted in decreased agonist potencies, although the effects on potency were not as large as obtained with the mutation to G (Figure 4 4A and C, Table 4 1). 4OH GTS 21, an 7

PAGE 88

88 selective partial agonist, activated 7W55V mutants as efficaciously as ACh activated the mutant (Figure 4 4C). The relatively conservative mutation of W55 to tyrosine or phenylalanine did not significantly affect ACh potency, as EC 50 values re mained near 30 M. However, the potencies of choline, 4OH GTS 21, and AR R17779 were all altered by these mutations (T able 4 1). Mutation of W55 to phenylalanine caused the EC 50 of 4OH GTS 21 to increase 6 fold (Figur e 4 4D) and mutation of W55 to tyrosi ne yielded mutant 7 receptors that did not give detectable responses to 4OH GTS 21. The low efficacy of 4OH GTS 21 for the W55G and W55Y 7 mutants is likely related to desensitization and/or channel block by 4OH GTS 21. Residual inhibition by 4OH GTS 21 in wild type 7 receptors was previously characterized [ 327 ] and was shown to represent a form of stabilized desensitization that is reversed b y the application of the t ype II PAM PNU 120596. Although 4OH GTS 21 was relatively ineffective at activating 7 W55G and W55Y mutant receptors, large currents occurred when 4OH GTS 21 was applied with PNU 120596 (not shown). These observations suggest that 4OH GTS 21 has access to the binding site in the 7 W55G and W55Y mutants, but that binding promotes PNU 120596 sensitive desensitization much more effectively than activation. Homologous mutations in the LBD of 4 2 receptors ( 2 W57) produced qualitatively different results from the mutations made in 7 Mutation to glycine greatly reduced ACh potency in 7 receptors, but increased ACh potency in 4 2W57 G mutants. Mutation of W55 to tyrosine in 7 receptors did not affect ACh potency, but ACh was twice as potent for 4 2W57 Y mutants as for wild type receptors. ACh also had greater potency for 4 2 W57F than for wild type 4 2 However, ACh had

PAGE 89

89 significantly lower potency for 4 2 W57A than for wild type 4 2. Neither choline nor AR R17779 activated any of the 4 2 receptors (Figure 4 5A E, Table 4 1). However, 4OH GTS 21 did activate four of the 4 2 W57 mutants and provided the most interesting differences amongst these W55 and W57 mutants of the 7 selective agonists tested. Note that maximal ACh responses of the 4 2 mut ants were approximately one tenth of the ma ximal responses in wild type receptors. While it is true that a selective compromise in ACh mediated activation would have the tendency to make the relative efficacies of the experimental agonists appear increase d, that effect would be manifested in the results of all experimental agonists normalized to the ACh responses. However, the data indicate that the increase in the relative efficacy of 4OH GTS 21 is many times larger than that of either choline or AR R177 79, which were both immeasurable because currents induced by these drugs were too small to be determined. Therefore, the increased activation of 4 2 mutants by 4OH GTS 21 is likely to represent a true potentiation of an activation mechanism potentially u nique to 4OH GTS 21 and related compounds. 4OH GTS 21 activated wild type 4 2 only about 2% and wild type 7 46% as well as ACh (Figure 4 2B and C). 4OH GTS 21 activated 4 2 W57A, glycine, phenylalanine and tyrosine but not 4 2 W57V, arginine, serine, or threonine mutant receptors. Interestingly, 4OH GTS 21 activated 7W55V mutants as well as ACh did. 4OH GTS 21 activated 4 2 W57A, phenylalanine a nd glycine to 60 70% of their ACh maxima, with lowest potency in 4 2 W57A and highest potency in 4 2W57Y and 4 2W57F mutants. Importantly, peak responses of 4 2 W57Y to 4OH GTS 21 were 2.5 fold greater than for ACh, while the homologous mutation in 7 decreased the relative efficacy of 4OH GTS 21 greatly.

PAGE 90

90 Mutation of W149 in B oth 7 and 4 2 Receptors R ed u ced Receptor Activation by B oth ACh and 7 Selective A gonists Responses of 7W149 mutant receptors were signifi cantly lower than those of wild type 7 receptors recorded the same number of days post injection. Somewhat surprisingly, the relatively conser vative mutations of 7W149 to F or Y disrupted receptor responses to ACh to levels below the limits of detection, while the non conservative mutations to A or G yielded receptors that were capable of producing measurable ACh induced currents. The 7W149V mutant receptors were also non responsive to ACh (Figure 4 6A). Of the mutant receptors that were non responsive to ACh, none were able to produce recordable currents in response to choline, 4OH GTS 21, or AR R17779, with the exception of 7W149F mutants, which reproducibly yielded small but measurable currents in response to choline (not shown). The fact that only two of the five 7W149 mutants were sufficiently functional for use in this study suggests that mutation introduced at position 149 may interf ere with receptor assembly or with conformational changes linked to channel opening. The mutations introduced at position 149 in 7 resulted in drastically reduced potencies of ACh, choline, and AR R17779 (Table 4 1). Mutation of 7W149 to glycine had pr ofound effects on receptor activation, resulting in 13 20 and 140 fold increases in EC 50 values over those seen in wild type receptors for ACh, choline, and AR R17779, respectively. However, neither 7W149A nor 7W149G mutant receptors had significantl y altered potency for 4OH GTS 21 compared to wild type. Compared to the maximum response of ACh in wild type receptors, the efficacy of choline fell 30% and the efficacy of 4OH GTS 21 rose 50% in 7W149G mutants (Figure 4 6C and D, Table 4 1).

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91 Alpha4W154 2 mutant receptors produced measurable ACh induced currents when mutations to A, F or W were introduced, while currents were undetectable when G was introduced at position 149, roughly the opposite of what was observed in 7W149 mutants (Figure 4 7A and B) This observation is suggestive of intrinsic differences between the 7 and 4 2 LBDs. There was no functional expression of 4W154G 2 or 4W154V 2 mutant receptors detected; these mutants were also non responsive to choline, 4OH GTS 21, and AR R17779 (n ot shown). Mutation of 4W154 to A resulted in a 7 fold increase in the EC 50 for ACh, while mutations to phenylalanine or tyrosine did not greatly alter ACh potency compared to wild type. Of the 4W154 2 mutants tested, none responded to choline, AR R17 779, or 4OH GTS 21 (Figure 4 7C, D, and E). In general, a loss of receptor function was observed as a result of any mutation introduced at the W149 position, at least for the agonists we tested and mutations we introduced. Taken together, these results s uggest the W55 position may better tolerate mutation than W149, and when mutated allows for major alterations in the receptor activation mechanisms, especially in regard to choline and 4OH GTS 21. Discussion Numerous mutational studies of 7W55 and 7W149 (and homologous residues in other nAChR subtypes) and other conserved aromatic residues within the aromatic box of the nAChR LBD generally lead one to believe that mutation of conserved aromatic residues results in reduced receptor functio n ality Building on evidence from a previous study, which showed that mutation of Y188 does not necessarily knock down

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92 receptor functionality for all agonists, the functional significance of the conserved 7W55, 2W57, 7W149 and 4W154 residues was inves tigated. Using unnatural amino acid substitutions in muscle type nAChR at positions W86, W149, W184, and $ W55/ # W57 with W derivatives containing various degrees of predicted cation interaction energies, W149 has been shown to establish primary interac tions with the quaternary ammonium group of ACh [ 107 396 ] Another implication of the data from Zhong et al 1998 was that W55 may not directly stabilize the quaternary group of ACh through cation & interactions, and the specific role of this residue in receptor activation remains an open question. In Torpedo receptors, mutation of $ W55 to leucine reportedly reduced ACh affinity 7,000 fold, while similar mutation of # W57 resulted in only a 20 fold reduction. Double mutant receptors ( $ W55L and # W57L) were reported to have reduced binding of many small agonists, including tetramethylammonium. Nicotine binding, however, was unaffected by the double mutant receptors [ 400 ] W55 was recently proposed to affect desensitization kinetics [ 403 ] However, results of this study are inconclusive since 7 currents were measured as peak responses [ 214 ] mutation of W55 affected agonist potency which in turn alters the response waveform [ 404 ] and macroscopic currents alone are insufficient to determi ne kinetics of desensitization [ 405 ] These data suggest that the conserved residues W149 and W55 have different functional significance in 7 nAChR. Mutations at the W55 residue appear to be relatively well tolerated since they provide subtype dependent effects on channel activation, suggesting they may be important determinants of subtype selective activation by certain agonists. For examp le, the 7 agonist 4OH GTS 21 lost efficacy in

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93 7W55 mutants relative to ACh while the same agonist gained efficacy, up to 300 fold compared to ACh, in some 4 2 W57 and 4Y190F 2 mutants. The observation that 4 2 receptors gain function with 4OH GTS 21 w hen W57 is mutated to smaller residues is consist ent with the idea that the wild type receptor has unfavorable steric clashes with this compound. In the case of the 7 receptor, the interactions of 4O H GTS 21 with W55 may be optimal such that in general, making this position smaller in size results in less favorable binding and/or function. In contrast to the variable effects of W55 mutations, W149 seems to serve a role that is universally important for receptor function regardless of agonist or recepto r subtype. However, if W149 were solely responsible for stabilizing the ligand, one would expect to see total loss of receptor activity when this residue became incapable of forming cation & interactions. Clearly this was not the case as non aromatic 7 W149 mutant receptors still responded to agonists, and mutation to other aromatic residues actually produced receptors that were non responsive to ACh. Other aromatic residues and non aromat ic residues found within the LBD probably help to stabilize and compensate for lost interactions resulting from mutation, or perhaps form an alternate set of interactions with ACh that are still capable of activating the receptor when the W149 is mutated. It seems likely that residues localized in the vicinity of the LBD may establish interactions with the ligand that place it in a position in which its ability to interact with the receptor and initiate changes in channel gating is regulated by conserved a mino acids such as W55, W149, or Y188. In the current study, 4OH GTS 21 is the only agonist tested which efficaciously activated certain 4 2 mutants while losing 7 efficacy. Likewise, in the previous study

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94 with Y188F mutants, anabaseine derivatives were the only 7 agonists tested that activated the mutant 4 2 receptors, with 4OH GTS 21 be ing the most efficacious by far [ 401 ] These observations raise qu estions regarding how 4OH GTS 21 activates nAChR. While it is possible that 4OH GTS 21 may fi t into the binding site of wild type 7 and 4 2 in different conformations, it is also likely that ligands such as ACh and 4OH GTS 21 form different types of int ermolecular interactions with residues in the protein, and thereby promote receptor activation in different ways. Investigations of AChBP bound with agonists and antagonists have led to the idea that channel opening and, possibly, ligand selectivity may o ccur through a conformation induced by the ligand which is due to the inherent flexibility of the binding site, allowing it to conform to the structural characteristics of the ligand [ 106 ] A potentially important observation made by Dr. Nicole Horenstein is that AC h choline, and AR R17779 all contain sp 3 hybridized ammonium nitrogen atoms while the nitrogen atom thought to be important in 4OH GTS 21 binding is sp 2 hybridized and flat. The difference in three dimensional structure between choline, AR R17779 and 4OH GTS 21 may underlie some of the unique observations made in this study regarding 4OH GTS 21. Adding insight to the results presented here is the report that serotonin activates the highly homologous mouse 5 HT 3 and C. elegans MOD 1 receptors through fo rmation of cation & interactions at different W residues, at the position homologous to W149 in 5HT 3 and at the position homologous to Y195 in MOD 1. In MOD 1 the residues at the positions homologous to 149 and 195 in 7 are Y and W, respectively [ 406 ] Mutation of these residues suggested that both receptors make specific contacts with serotonin that regulate channel gating, but those specific contacts depend on the

PAGE 95

95 nature of the binding site These observations could be consistent with either hypothesis discussed above. Evolutionary pressures may have allowed some flexibility in the ability of conserved aromatic residues to act differentially in different receptors, or serotonin may be acco mmodating to the distinct binding domains, finding alternate ways to establish interactions that lead to receptor activation.

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96 Figure 4 1. Multiple sequence alignment and hypothetical localization of 4W154, 2W57, 7W55, and 7W149. A) Closeup of the LBD from the crystal structure of AChBP isolated from L. stagnalis (PDB ID: 1i9B; [ 118 ] ) Numbering of resid ues correspond to human 7 numbering. The image was produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco [ 407 ] B) Alignment of human nAChR sequences wit h sample sequences from chick, C. elegans, and zebrafish show a high degree of conservation at both W residues throughout the nicotinic family and many species. Curiously, these tryptophans are also conserved in the 5 and 3 subunits, which have been proposed to occupy the accessory subunit position and not contribute directly to the agonist binding domain [ 132 ] Sequence alignments were generated using ClustalW at www.ebi.ac.uk/Tools/clustalw2/index.html [ 408 ]

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97 Figure 4 2. Concentration response relationships of wild type 7 and 4 2 receptors to ACh, choline, 4OH GTS 21, and AR R17779. A) Chemical structures of the agonists used in this study. B) Net charge responses o f wild type 7. C) Peak responses of wild type 4 2. Each data point represents the mean SEM of at least four oocytes. For consistency the single site hill equation was used to fit the 4 2 curve since the single site model provided the best fit for most 4 2 mutants in this study. There were no significant differences in chi square or R values between fits by the single site or biphasic models

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98 Figure 4 3. Functional responses of human 7W55 and human 4 2W57 mutant receptors relative to ACh in d uced maximum responses in wild type. A) Maximum net charge responses of 7W55 mutant receptors relative to the ACh induced maxi mum net charge response in wild type 7, represented as a value of 1. and ** denotes statistically significant differences in maximal fu nctional responses between wild type and mutant receptors with p<0.05 and p<0.01, respectively. B) Representative data traces of responses by 7 wild type and 7W55 mutants to 300 M ACh. C) Maximum peak responses of 4 2W57 mutants relative to the ACh induce d maximum peak response in wild type 4 2, represented as a value of 1. and ** denotes p<0.05 and p<0.01, respectively. D) Representative data traces of 4 2W57 mutant receptors in response to 30 M ACh. Maximum responses for mutant recept ors compared to wild type were cal culated by averaging responses SEM of at least four oocytes to the same co ncentration of ACh on both wild type and mutant receptors injected the same day, with the same amount of RNA from the same harvest of oocytes. Av eraged responses were divided by the percent of ACh maximum for that concentration on a fitted ACh concentration response curve to find the maximum theoretical response, and then divided by the calculate d maximum response for the wild type receptor for the comparison.

PAGE 99

99 Figure 4 4. Concentration response relationship of 7W55 mutant receptors to ACh, choline, 4OH GTS 21, and AR R17779. A) Net charge responses of 7W55A mutants. B) Net charge responses of 7W55G mutants. Note that this mutant did not res pond to 4OH GTS 21 at the concentrations tested. Due to concerns related to channel block and osmotic effects, the highest choline concentration tested was 10 mM. C) Net charge responses of 7W55V mutants. D) Net charge responses of 7W55F mutants. E) Net charge responses of 7W55Y mutants. Note the low efficacy of 4OH GTS 21. Responses of wild type 7 are presented in Figure 4 2B. Data were measured relative to control ACh responses and then expressed relative to the maximum ACh response for each part icular receptor type. Each point represents the mean SEM of at least four oocytes.

PAGE 100

100 Figure 4 5. Concentration response relationships of 4 2W57 mutants to ACh, choline, 4OH GTS 21, and AR R17779. A) Peak responses of 4 2W57A mutant receptors. B) Peak responses of 4 2W57G mutant receptors. C) Peak responses of 4 2W57V mutant receptors. D) Peak responses of 4 2W57F mutant receptors. E) Peak responses of 4 2W57Y mutant receptors. Note that 4 2W57A, 4 2W57G, 4 2W57F, and 4 2W57Y mutants re sponded to 4OH GTS 21. Responses to choline and AR R17779 were below the limits of detection of all 4 2 receptors. Data were measured relative to control ACh responses and then expressed relative to the maximum ACh response for each particular receptor type. Each point represents the mean S EM of at least four oocyt es. Peak responses by wild type 4 2 are presented in Figure 4 2C.

PAGE 101

101 Figure 4 6. Concentration response relationships of 7W149 mutants to ACh, choline, 4OH GTS 21, and AR R17779. A) M aximum ACh induced net charge responses of 7W149 mutants compared to maximum net charge response of ACh in wild type 7. ** denotes p<0.01. B) Representative data traces of 7W149 mutants in response to 300 M ACh. C) Net charge responses of 7W149A mu tant receptors. D) Net charge responses of 7W149G mutant receptors. Each data point represents the mean SEM of at least f our oocytes. Responses of wild type 7 to these agonists are presented in Figure 4 2B.

PAGE 102

102 Figure 4 7. Concentration response r elationships of 4W154 2 mutants to ACh, choline, 4OH GTS 21, and AR R17779. A) Maximum ACh induced peak responses of 7W149 mutants compared to the maxim um peak response of ACh in wild type 7. ** denotes p<0.01. B) Representative data traces of 4W154 2 mutants in response to 30 M ACh. C) Peak responses of 4W154A 2 mutants. D) Peak responses of 4W154F 2 mutants. E) Peak responses of 4W154A 2 mutants. Each data point represents the mean SEM of at least four o ocytes. Peak responses by wild type 4 2 are represented in Figure 4 2C.

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103 Table 4 1. EC 50 and I max values of ACh, Choline, 4OH GTS 21, and AR R17779 in wild type and mutant 7 and 4 2 receptors I max SEM relative I max SEM relative Drug Receptor EC 50 SEM to ACh maximum to ACh maximum ACh 7 30.6 3.36 1 1 7W55A 84.8 5.79 1 2.5 0.40 7W55F 32.7 2.70 1 0.52 0.10 7W55G 675 6.26 1 2.4 0.25 7W55V 84.2 2.53 1 1.2 0.12 7W55Y a 30.4 5.03 1 0.57 0.13 7W149A 194 9.73 1 0.15 0.03 7W149G 400 19.3 1 0.17 0.03 4 2 76.0 20.2 1 1 4 2 W57A 468 59.3 1 0.094 0.011 4 2W57F 33.3 4.03 1 0.036 0.017 4 2W57G 47.4 10.9 1 0.085 0.015 4 2W57V 87.0 10.4 1 0.050 0.0073 4 2W57Y 16.2 1.60 1 0.076 0.033 4W154A 2 526 28.1 1 0.40 0.04 4W154F 2 118 6.07 1 0.39 0.06 4W154Y 2 81. 1 3.44 1 0.04 0.00 Choline 7 304 21.8 0.90 0.02 0.90 0.02 7W55A 949 210 0.95 0.05 2.34 0.12 7W55F 502 71.8 1.4 0.0 7 0.73 0.04 7W55G 4120 63.4 3.1 0.03 7.47 0.07 7W55V 570 12.4 1.1 0.01 1.38 0.01 7W55Y 228 13.2 0.84 0.01 0. 48 0.01 7W149A 2490 126 0.89 0.03 0.134 0.005 7W149G 68 70 1440 0.61 0.07 0.104 0.012 4OH GTS 21 7 3.00 0.26 0.46 0.01 0.46 0.01 7W55A 8.40 0.73 0.33 0.01 0.82 0.02 7W55F 18.9 1.93 0.45 0.02 0.23 0.01 7W55G NA b NA b NA b 7W55V 7.80 0.82 0.94 0.03 0.54 0.02 7W55Y NA b NA b NA b 7W149A 4.49 1.39 0.28 0.04 0.042 0.006 7W149G 4.43 0.50 0.76 0.08 0.130 0.014 4 2 NA b 0.01 0.00 0.01 0.00 4 2 W57A 2.56 0.27 0.71 0.02 0.067 0.0019 4 2W57F 0.66 0.22 0.62 0.05 0.022 0.0018 4 2W57G 1.16 0.20 0.74 0.04 0.063 0.0033 4 2W57V 3.00 0.14 0.07 0. 01 0.0035 0.0005 4 2W57Y 0.65 0.09 2.7 0.08 0.19 0.006 AR R17779 7 3.60 0.73 1.05 0.05 1.05 0.05 7W55A 32.4 1.69 0.96 0.02 2.36 0.05 7W55F 82.3 2.51 1.02 0.01 0.53 0.005 7W55G 332 0.68 0.99 0.01 2.36 0.02 7W55V 93.9 5.91 1.05 0.04 1.26 0.05 7W55Y 86.8 42.1 0.90 0.16 0.51 0.09 7W149A 119 3.99 1.07 0.14 0.16 0.021 7W149G 506 72.3 0.97 0.09 0.17 0.015 a Responses were normalized to 30 M ACh b Res ponses were below the limits of detection Note: The average values for each data point was 7 and n values ranged from 4 to 18

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104 CHAPTER 5 THE EFFECTIVE OPENIN G OF NICOTINIC ACETY LCHOLINE RECEPTORS W ITH SINGLE AGONIST BINDI NG SITES Introduction As ex plained in Chapter 2, the nAChR LBD is formed by the interface of two protein subunits; the primary surface is formed by an subunit, which contains several other key elements in addition to the adjacent cysteines of a subdomain identified as the C loop [ 108 ] Th e complementary face of an agonist binding site is formed by a non subunit in heteromeric nAChRs and by an subunit in homomeric nAChRs. Subunits like 7 are able to contribute to five agonist binding sites at both primary and complementary interface s urfaces [ 133 ] while heteromeric r eceptors, which require non subunits for the complementary side of the binding site, are limited to two agonist binding sites. Acetylcholine is a nearly perfect molecule for fast and transient synaptic signaling at sites like the neuromuscular junct ion, being rapidly released and efficiently hydrolyzed. However, nicotinic signaling appears to be fundamentally different in the brain, where rhythmic ACh release occurs diffusely, rather than at focused synaptic sites, and a primary role of nAChRs in the brain is to modulate neuronal excitability and the release of other neurotransmitters [ 163 187 ] Due to esterases, extracellular concentrations of diffusely released ACh are expected to be low. While choline is ubiquitous in the brain and body, concentrations are still well below the EC 50 for acute activation of 7 [ 214 ] even under conditions of trauma when choline concentrations reach 100 M [ 219 409 ] In addition, responses of 7 receptors to high ACh concentrations are very limited [ 214 229 ] which raises the question of whether 7

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105 nAChR may function effectively under conditions of low fractional occupancy of the agonist binding sites. Single channel studies of muscle type nAChR have associated brief openings o bserved at low agonist concentrations with monoliganded receptors [ 69 410 412 ] supporting the hypothes is that the brief openings characteristic of 7 may also arise from the binding of single agonist molecules. Here, the functional significance of the multiple agonist binding sites in heteromeric muscle type and homomeric 7 forms of nAChR is investigated utilizing the L119C mutation ( 7 numbering) which is located on the complementary face of the agonist binding site across from the C loop (see Figure 5 1A), together with sulfhydryl modification at that site to achieve varying levels of conditional binding site modification [ 128 ] Acetylcholin e insensitive 7 Y188F subunits [ 413 ] co expressed with wild type 7 subunits in different ratios are also used to provide a complimentary approach. The data are consistent with previous reports that heteromeric muscle type receptors and homomeric Cys loop receptors can activate with levels of submaximal agonist occupancy [ 412 414 420 ] The data offer the additional insight that strong activation of muscle type and 7 nAChR may be achieved under conditions of agonist saturation at individual binding sites. Results The sensitivity of 7L119C mutant receptors was discovered by Dr. Roger Papke and the experiments involving oocytes were performed before I was a member of the lab group. My contributions to this project were the patch clamp experiments and their integration with the oocyte data, which proved necessary in publishing and understanding this earlier work.

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106 Identification of the 7L119C Mutation as a Tool to Study nAChR Binding S ites The 7 receptor contains a free cysteine residue at position 116. In order to prevent non specific modification and/or potential disulfide formations between the single free cysteine at position 116 and the introduced cysteine, a cysteine null pseudo wild type C116S background was used. Responses of pseudo wild type 7 C116S receptors to EC 50 concentrations of ACh, tetramethyl ammonium, quinuclidine, and 4OH GTS 21 are not si gnificantly different from wild type, and are unaffected by application of MTSEA [ 128 ] A similar pseudo wild type background was used in other studies that introduced cysteine mutations into 7 [ 421 ] The L119C mutation was identified as an effective tool for the investigation of nAChR binding sites since receptors containing this mutation responded normally to ACh, tetramethyl ammonium quinuclidine, and 4OH GTS 21 until treated with MTSEA or any of the three other cationic sulfhydryl reagents applied, after which agonist induced responses were nearly completely abolished [ 128 ] The near 100% re duction in response to 300 M ACh that is typical when 2 mM MTSEA is applied for 60 s to 7C116S/L119C mutant receptors expressed in oocytes is shown in Figure 5 1B. The degree of inhibition is not significantly dependent on the ACh concentration used to evoke the responses. In the specific experiment illustrated, net charge responses to 300 M ACh were reduced by 99.7 0.1%, and responses to 3 mM ACh were reduced to a similar extent (95.9 2.0%, data not shown). Effects of 7 L119C Ratios in M ixed 7 W ild Type/Mutant H eteromers The purpose of the following experiments was to test the hypothesis that 7 receptors may be activated, even if the receptors have a reduced number of activatible

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107 binding sites. Xenopus oocytes were injected with RNA coding fo r 7 C116S (pseudo wild type) subunits and the MTSEA sensitive 7 C116S/L119C subunits in varying ratios. The possible subunit combinations and likely distributions of those combinations as a probabilistic function of the RNA ratios, assuming equal expressi on and assembly of the wild type and mutant subunits, are shown in Figure 5 2A. With the highest ratio (5:1) of 7 C116S/L119C to 7 C116S, less than 4% of the receptors would be predicted to have more than two MTSEA insensitive binding sites, and 40% would be predicted to be fully MTSEA sensitive. An attempt was made to verify the expression of receptors containing 7 L119C mutant subunits by comparison of radiolabeled b ungarotoxin binding before and after MTSEA treatment. However, the 7 C116S and 7 L119C mutations appeared to disrupt the binding of the competitive antagonist, and such experiments were not possible to pursue. Comparison of the magnitude of non normalized functional responses from oocytes to the same concentration of ACh (300 M) that were injected the same day, with the same amount of RNA from the same harvest of oocytes, revealed equivalent responses among the 7 C116S/L119C, 1:1, 3:1, 5:1 groups. The functional responses of the oocytes injected with 7 C116S alone were 3 fold greater (dat a not shown, n $ 5 oocytes for each group). Control responses to 300 M ACh were recorded for each cell in the three injection sets prior to MTSEA treatment (Figure 5 2B). After treating the oocytes with MTSEA (2 mM, 60s), oocytes were tested with a ra nge of ACh concentrations from 30 M to 1 mM. The average net charge data are shown in Figure 5 2C (see also Table 5 1), normalized to the 300 M ACh net charge responses obtained prior to the MTSEA treatment. The most obvious effect of the MTSEA treatme nt was on the responses to

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108 low ACh concentration in the oocytes injected with the highest fraction of MTSEA sensitive mutants. In the control (pre MTSEA) condition, 300 M ACh was sufficient to produce a maximal net charge response. Responses of the oocy tes injected at ratios of 1:1 and 3:1 showed no significant differences in function following MTSEA treatment (p > 0.05). Only the oocytes injected at the 5:1 ratio showed a significant decrease in the 300 M ACh evoked responses (p <0.05) following MTSEA treatment; however, this average decrease of 33% was less than the percentage of receptors predicted to be fully sensitive to MTSEA (40%, Figure 5 2A). While untreated 7 receptors (not shown) and the treated receptors injected at 1:1 (Figure 5 2C) showed no increase in net charge from 300 M to 1 mM ACh, the average responses of the 5:1 injected oocytes to 1 mM ACh increased to the extent that responses to 1 mM ACh were not significantly different from the pretreatment 300 M control responses at the p < 0.05 level. One possible explanation is that 7 receptors with one or two functional binding sites may be less affected by the rapid concentration dependent desensitiza tion that is characteristic of 7 and, therefore, better able to respond to high concentrations of agonist. Effects of ACh Insensitive Mutant Ratios in M ixed 7 W ild Type/Mutant H eteromers A mutation in the primary face of the 7 ACh binding site (Y188F) that produces a 45 fold reduction in ACh potency (ACh EC 50 shifted from 33 4 M for wild type to 1500 164 M for 7 Y188F) without any significant effect on the potency of the 7 selective partial agonist 4OH GTS 21 (4OH GTS 21 EC 50 14 1 M for wild type and 14 2 M for 7 Y188F) was previously reported [ 413 ] As shown in Figure 5 3A, for wild type 7 the ratio of the 300 M 4OH GTS 21 evoked net charg e responses to 300 M ACh evoked responses were 0.57 0.04. In contrast, for 7 Y188F receptors, the ratios of

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109 the 300 M 4OH GTS 21 evoked responses to 300 M ACh evoked responses were 4.0 0.6. Acetylcholine sensitive wild type 7 and ACh insensitive 7 Y188F were co expressed in Xenopus oocytes at varying ratios of RNA, similar to what was done with the L119C mutant (Figure 5 2B and C). The response evoked by 4OH GTS 21 on 7 Y188F receptors is consistent with efficient 7 Y188F subunit expression and a ssembly in oocytes [ 413 ] The mutant and wild type subunits responded alike to 300 M 4OH GTS 21, but wild type subunits were required to generate responses to 300 M ACh. However, if only single subunits were required to activate a receptor, then even under the condition when 7 Y188F was injected at a 5:1 ratio to wild type 7 the net charge responses to ACh should remain relatively high since 60% of the r eceptors would have at least one ACh sensitive wild type subunit. As shown in Figure 5 3B, the sensitivity of the wild type receptor to ACh was retained well, even under the 5:1 injection condition, when it would be predicted that very few of the receptor s w ould have more than 1 or 2 wild type subunits. The shift in 4OH GTS 21 to ACh response ratios was no more than would have been expected from the prediction that 40% receptors would contain only ACh insensitive subunits. Effects of Mutations H omologous to 7L119C in N on Subunits of Muscle Type R eceptors Since modeling of the homomeric 7 subunits places the L119 residue in the complementary face of the agonist binding site (Figure 5 1A), this site is expected to form the specialized domains correspondi ng to those of the non subunits in heteromeric nAChR. Consistent with this prediction, MTSEA treatment reduced responses to high (1 mM) and low (30 M) ACh concentrations by more than 95% when the mutation homologous to 7 L119C was placed in the 2 subu nits of 4 2 and 3 2

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110 receptors, while little MTSEA dependent effects were observed when the homologous mutation was placed in the subunits [ 128 ] While the placement of the modifiable L119C residue in 7 receptors and the modifiable 2 L121C in 4 2 and 3 2 receptors impacts all potential binding sites of these receptors and produces equally profound reduction in function following MTSEA treatment, the effect of the homologous mutation in subunits of heteromeric muscle type receptors would be expecte d to depend on the specific subunit(s) in which the mutation was placed since the 1 subunits are paired with different non subunits ( # and $ or % ), which contribute different complementary faces to the two agonist binding sites. While MTSEA treatment p roduced no significant decreases in t he ACh evoked responses of wild type 1 1 %# or 1 1 $# receptors across a range of ACh concentrations from 1 M to 1 mM (see Figure 5 4 and Table 5 2), receptors with mutations in both % and # had large reductions in the ir responses to both low and high concentrations of ACh (Figure 5 4). Peak current and net charge responses of the double mutants ( 1 1 % L119C # L121C), to 30 M ACh were reduced 93 1 and 96 1 %, respectively, and peak current and net charge responses to 1 mM ACh were reduced by 82 6 and 91 3 %, respectively. However, if mutations were placed in only one of the two non subunits that contribute to agonist binding sites ( 1 1 $# L121C, 1 1 % L119C # or 1 1 %# L121C), MTSEA treatment produced large decrea ses (p<0.01) in the responses evoked by 30 M ACh with less effect on the 1 mM ACh evoked peak current responses for either the 1 1 $# L121C or 1 1 % L119C # receptors. Although peak current responses to 1 mM ACh for the 1 1 %# L121C receptors were decreased by MTSEA treatment (p<0.01), the effect on 1 mM ACh responses (30 7% decrease) was much less (p<0.0001) than that on 30 M ACh

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111 responses (74 4% decrease). Note that although the peak amplitude of the responses evoked from 1 1 $# L121C and 1 1 % L119C # r eceptors by 1 mM were not decreased by the MTSEA treatment, the net charge values of the 1 mM ACh evoked responses on all single subunit mutants were affected because the MTSEA treatment resulted in currents with significantly (p<0.05) faster 90% 10% decay times. This would be consistent with a decreased ability of the treated oocytes to respond to the lower concentrations of ACh during the washout period. The ACh concentration response curves for the peak current responses of the muscle type receptors c ontaining MTSEA sensitive mutations are shown in Figure 5 5, and the fit I max and EC 50 values are shown in Table 5 2. In addition to obtaining responses to varying concentrations of ACh, multiple responses to ACh at the control concentration of 30 M were obtained from each cell before and after MTSEA. These are shown in the right hand column of plots in Figure 5 5. Note that there was some recovery in the size of the 30 M control responses after the MTSEA treatments. Given that the MTSEA modification results in a covalent bond, reversibility of the effect seems unlikely. The response reversibility could have represented the insertion of new receptors during the course of the experiment. Muscle type receptors express better than any other nAChR subtyp e, and oocytes must be studied immediately on the day after injection or else currents are too large for effective voltage clamping. Another possibility is that the receptors with one unmodified binding site show less of an effect of progressive desensiti zation with repeated agonist applications. To further investigate the MTSEA resistant activation of muscle type receptors with one unperturbed binding site, single channel patch clamp exper iments were

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112 performed with wild type and mutant 1 1 $# receptors tr ansientl y transfected in mammalian BOSC 23 cells. The fetal 1 1 $# version of the muscle type receptor was the focus of these experiments since it is the subject of an extensive literature, due to its expression in BC 3 H1 cells (see for example [ 422 425 ] Two main types of analyses were performed on the single channel data: (1) comparison of post MTSEA treatment peak current and NP open measurements with the pre MTSE A measurements obtained from the same patch in response to 1 mM ACh; and (2) fitting of burst duration histograms from untreated and treated receptors. At a holding potential of 70 mV, the single channel amplitudes before and after MTSEA treatment (5 mM 60 s) o f wild type receptors were 2.71 0.02 pA and 2.69 0.01 pA, respectively, of 1 1 $# L121C mutant receptors were 2.64 0.05 pA and 2.63 0.03 pA, respectively, and of 1 1 $ L119C # L121C double mutant receptors were 2.74 0.05 pA and 2.70 0.06 pA, respectively. The single channel amplitude with 10 nM ACh concentration at 70 mV was 3.13 0.02 pA. The smaller apparent single channel amplitude observed with high concentrations of ACh would be consistent with an effect of brief episodes of channel block by agonist, limiting the detection of full amplitude events [ 426 ] The single channel slope conductance of 1 1 $# L121C receptors was 35.5 1.2 pS and the reversal potential was 4.4 2.0 mV ( 80 mV to 80 mV, n = 3, data not shown). These values are i n agreement with previously published studies on fetal muscle type nAChR [ 94 427 428 ] Figure 5 6A shows traces from outside out patches under the experimental protocol, which consisted of an ~80 s initial ACh application, followed by either 5 mM MTSEA or external saline application for 60 s, and ended with a follow up ACh

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113 application. External saline instead of 5 mM MTSEA was applied to some patches expressing 1 1 $# L121C to allow channel rundown and/or desensitization that may occur independently of any MTSEA dependent effects to be measured. Figure 5 6B summarizes the post MTSEA treatment versus pre MTSEA comparisons of both transient peak currents and NP open with 1 mM ACh, normalized to the average values from 8 rundown/desensitization control patches. Despite consistent experimental setup, the pa tch to patch variability of the peak current and NP open measurements was higher than expected for unknown reasons that probably reflect the unstable and fragile nature of outside out patches. No attempt was made to identify or eliminate outliers. There w ere no correlations between post /pre MTSEA treatment peak and NP open measurements and transfection dates, recording dates, lower limit of N in the patch, single channel amplitudes, or 10% 90% rise times. The non normalized average, SEM range, and median of values from at least 8 replicates of each condition, including the rundown controls, are reported in Table 5 3. Peak and NP open measurements of wild type receptors were least affected by MTSEA treatment (5 mM, 60 s) with post/pre MTSEA treatment value s of 1.1 0.1 and 0.83 0.09, respectively. Receptors with one MTSEA sensitive binding site had post/pre treatment peak current and NP open values of 0.74 0.08 and 0.65 0.06, respectively. There was a much greater reduction in both peak and NP open v alues when receptors contained two MTSEA sensitive binding sites, with post/pre treatment values of 0.072 0.003 and 0.14 0.02, respectively. Figure 5 7 presents single chan nel currents obtained from wild type and 1 1 $# L121C receptors at 1 mM ACh before and after MTSEA treatment, responses from untreated 1 1 $# L121C receptors at 10 nM ACh, and fit burst duration histograms

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114 corresponding to each condition. The fit time constants are listed in Table 5 4. Burst dura tions, rather than apparent open duration histograms are shown since open channel times are greatly affected by bandwidth limitations, such as missed brief closed intervals and ambiguities associated with idealization, whereas burst durations are much less affected by these potential confounders and are, therefore, a more reliable measure of channel opening behavio r. The burst durations of wild type 1 1 $# receptors were unaffected by MTSEA treatment, while the burst durations of 1 1 $# L121C recepto rs were not different from wild type receptors until treated with MTSEA. The number of components required to generate the best fit of the 1 1 $# L121C bur st duration histogram after MTSEA increased from two to three, and most bursts consisted of brief, isolated openings, but some longer bursts remained. The proportion of total bursts of duration less than ~ 2 ms increased from 0.26 0.04 before MTSEA trea tment to 0.81 0.03 after MTSEA treatment. The longest time constant following MTSEA treatment was equivalent to the long time constant measured on non treated patches (12.6 0.2 ms versus 12.9 0.05 ms). The frequency (bursts/s) of bursts appeared to increase after MTSEA treatment by a factor of 2.78 0.52. In the case of 1 1 $# L121C receptors post MTSEA treatment, "bursts" were usually brief and isolated events. Recordings from !"$# L121 mutants were also obtained without MTSEA treatment at low ACh concentrations (10 nM), where many of the observed openings were likely to arise from singly occupied receptors [ 69 412 ] The proportion of bursts corresponding to the brief time constant (0.26 0.07 ms) was 0.43 0.02, and the long time constant at 10 nM ACh was more brief than the long time constant at 1 mM ACh (4.66 0.04 ms versus 12.9 0.05 ms).

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115 Dis cussion Other studies have investigated the relationship between available binding sites and agonist evoked responses in muscle type receptors [ 100 416 ] and in an 7/5 HT3 chimeric receptor [ 420 ] Sine and Taylor 1980 estimated the fractional blockade of binding sites with cobratoxin and then measured ion flux in toxin bound vesicles from BC3H1 cells (fetal receptor). The conclusion was that only receptors with two free agonist binding sites could be activated. However, Groebe et al 1995 reported that application of 500 nM conotoxin M1 to BC3H1 cells almost complete ly inhibits agonist evoked responses even though the 500 nM concentration of conotoxin M1 theoretically results in < 3% occupation of the low affinity binding site ( $ ) by the toxin on the BC3H1 nAChR [ 429 ] In addition, Hansen et al ., 200 5 solved crystal stru ctures for three states in the agonist binding site of the AChBP one of which was not part of the minimal model for the nAChR (closed, open, and desensitized states). This state was induced by the binding of cobratoxin, but not by the binding of relatively small competitive antagonists like MLA. In this state, the C loop extended in the opposite direction from which it presumably moves during the activation process [ 106 ] Therefore, it is possible that i n the origina l Sine and Taylor 1980 experiments the large conformational change produced by the binding of just one cobratoxin molecule was sufficient to render the entire channel una ble to gate. Jha and Auerbach 2010 used mutations at position W149 in adult recept ors to produce binding sites with reduced sensitivity to ACh, with the conclusions being that a single agonist binding site can activate the receptor, but with much less efficiency than two binding sites (equilibrium gating constants: E 1 ACh ( 4.3 x 10 3 vs E 2 ACh ( 28). However, while expression of

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116 W149 mutant subunits resulted in receptors with two mutant binding sites and greatly reduced responses to ACh, co expression of W149 mutant subunits with wild type subunits to produce receptors with single functional binding sites resulted in mixed pop ulations of r eceptors consisting of all wild type subunits, single W149 mutant subunits, and two mutant W149 mutant subunits. Rayes et al ., 2009 used an 7/5 HT 3 receptor chimera with subunits containing 7Y190T and/or 7W55T mutations to reduce ACh sen sitivity. In addition, the mutant subunits contained reporter mutations in the 5 HT 3 sequence that altered unitary channel conductance so that receptor subunit combinations could be identified. The conclusions of this study were that 7/5 HT 3 chimeric re ceptors activated partially when receptors contained fewe r than three wild type s ubunits, or when the three wild type subunits were located at adjacent subunit interfaces. In addition, the authors concluded that receptors with fewer wild type binding site s experienced less desensitization following strong stimulation by ACh. Unfortunately, the 7/5 HT 3 chimera is a "man made" receptor with a much higher open probability than native 7 nAChR, and so it is unclear to what extent the conclusions of this study may be applied to 7. While the approach used here is not without its own limitations, it provides the significant strength over the loss of function mutation approach that responses can be recorded from the same receptor population before and after binding site modification. Notwi thstanding the Sine and Taylor 1980 experiments, the general c onsensus from early single channel recordings of muscle type receptors (see below) and experiments utilizing loss of function mutations suggest that a single agonist binding site is sufficient to open the nAChR channel, but that openings from a single bind ing site have a low P open relative to that of openings arising

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117 from multiple binding sites. The results presented here are not inconsistent with this observation given that responses to ACh were reduced at low concentrations after binding site modificatio n. Early single channel studies of muscle type nAChR noted that the component of brief openings at low agonist concentrations behaved as if it arose from monoliganded receptors. The ratio of total events corresponding to brief, isolated openings (fast ti me constant) versus longer lived openings (slow time constant) that were attributed to di liganded receptors was relatively high at low agonist concentrations, but decreased as agonist concentrations increased [ 69 410 412 425 427 ] Interestingly, however, a component of short lived openings persisted even at high agonist concentrations, accounting for approximately 10% of all apparent channel openings [ 412 427 ] Prior to performing the patch clamp experiments, only brief, isolated openings were hypothesized to occur f ollowing MTSEA treatment of # L121C receptors, with the frequency of such events increasing as agonist concentration increased. The majority (81 %) of bursts were indeed brief ( % 2 ms); however, some longer bursts (~12.6 ms) were also observed. This obser vation, together with previous observations that brief, isolated single channel openings persist at high agonist concentrations [ 412 ] suggest the interesting possibility that both short and longer lived channel activations may arise from either mono or di liganded receptors, with a single liganded binding site opening with greatest probability to the shorter lived opening and the diliganded receptor opening to the longer lived open state with greater probability. In general, the data support the interpretation that conversion to the desensitized state occurs as in relation to the total time spent in the open state. Individual openings from singly liganded

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118 receptors may with higher likelihood be brief; but nonetheless, if entry into desensitized states depends strictly on time in the open state, then such receptors would generate the same net amou nt of current before desensitizing as diliganded receptors. Ideally, MTSEA modification occurred to completion at all "L119C" sites, producing binding sites with no affinity for ACh, and with no effect on channel gating itself. The observation that so me currents were recorded after MTSEA was applied to the 1 1 $ L121C # L119C double mutants in both two electrode voltage clamp and patch clamp experiments is problematic. Does the MTSEA modification only partially reduce the binding site sensitivity to ACh or were some receptors left unmodified? Unfortunately neither question is straightforward to answer. In a study evaluating residues that contribute to b ungarotoxin binding in muscle type receptors, Sine 1997 demonstrated that MTSET modification of rece ptors containing # L121C, $ L119C, or % L119C residues resulted in a 50% decrease in bungarotoxin binding, as would be expected if the receptors contained only one modified binding site and modification completely prevented b ungarotoxin binding. Furtherm ore, analysis of the binding site selective antagonists dimethyl d tubocurarine and conotoxin M1 confirmed that the effect was due to specific modification at the selected binding site. Electrostatic repulsion, rather than effects on channel conformatio n, was hypothesized to be responsible for the disruption of bungarotoxin binding following MTSET modification [ 430 ] In addition, a recently constructed homology model of MTSET modified 7L119C suggests that the modification p laces a hard positive charge in close proximity to where ACh is expected to bind. Because MTSET differs from MTSEA only in the substitution of methyl groups for hydrogens on the quaternary nitrogen of the sulfhydryl reagent, the

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119 model for MTSEA modified r eceptors is expected to be equivalent [ 128 ] From these observations and given that ACh contains a positively charged ammonium group, it seems most likely that the residual current by the double mutants was due to a small fraction of incompletely modif ied receptors. Could the modification have spared some or all of the binding site affinity for ACh, primarily affecting the ability of the binding site to initiate the gating cascade once ACh bound? Protocols using 7 C116S/L119C mutants, MTSEA, and the PAM PNU 120596, which converts some desensitized states into conducting states, suggest that MTSEA modification may stabilize mutant 7 receptors in PNU 120596 sensitive desensitized states since application of PNU 120 596 alone following MTSEA modification produces activation. Importantly, MTSEA modified receptors remain insensitive to ACh, even after they have been primed by the powerful allosteric modulator PNU 120596 [ 380 ] However, although the receptor modeling and data favor the position that ACh is excluded from the ligand binding site, the modification by MTSEA could conceivably mimic a permanently bound weak partial a gonist, potentially increasing the ability of the unmodified binding site(s) to activate more readily upon binding of the full agonist ACh. Since the proportion of the total synaptic current from monoliganded muscle type receptors under normal physiologic al conditions is likely negligible, the data are arguably most interesting in their possible application to neuronal nAChRs in the brain, where evidence for nicotinic synaptic transmission is slim, and agonist concentrations are expected to be low. In fac t, some have wondered whether nAChRs in the brain even see ACh at all [ 431 ] The data show that Xenopus oocytes injected with a high percentage of 7 L119C mutant RNA, and therefore likely to have a reduced number of

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120 available agonist binding sites following MTSEA treatments, can give responses to high agonist concentrations after MTSEA treatment that are comparable to the responses of receptors with all binding sites intact obtained at lower concentrations of agonist. Likewise, combinations of wild type and 7 Y188F receptors likely to have very few A Ch sensitive wild type subunits, nonetheless respond well to ACh. If submaximal occupancy by agonist i s sufficient to activate heteromeric and homomeric receptors, what is the role of the additional binding sites? Especially in the case of 7 the functional consequence of having multiple ACh binding sites may be sensitivity to low levels of agonist, rath er than to require high levels of occupancy for activation. High levels of agonist occupancy only appear to promote desensitization, or at least to provide sufficient activation during the process of achieving high levels of agonist site occupancy for the receptors to become desensitized. When challenged with a high concentration of agonist, 7 receptors open with highest probability during the leading edge of the solution exchange, when only a fraction of the agonist binding sites would be occupied [ 2 13 214 218 229 432 ] after which t he receptors are predominantly closed or desensitized. This is true whether recordings are made on a slow time scale in oocytes [ 213 ] or on a rapid time scale with dissociated neurons [ 432 ] and this is so pronounced that with the nor mal experimental protocol the time integrated (i.e. net charge) response to 3 mM ACh shows no significant increase over that evoked by 100 M ACh. The rapid desensitization of 7 may be a protective mechanism against cytotoxicity induced by excess entry of calcium. The nature of the desensitized state appears to be rather different for heteromeric and homomeric receptors. For heteromeric receptors, desensitization is associated with

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121 an approximately thousand fold increase in the apparent affinity of th e agonist binding site for ACh, while in 7 receptors there does not appear to be any more than a tenfold increase in agonist affinity with desensitization [ 432 ] T he conversion of heteromeric receptors to a high affinity, desensitized state means they will be likely to retain agonist at the binding sites or rebind agonist even as agonist concentration decreases. This may have the effect of stabilizing the desensiti zed state and slowing recovery. In contrast, agonist will readily dissociate from homomeric 7 receptors, so that in the presence of low concentrations of agonist, the receptors may easily cycle between activation and desensitization and generate a signif icant time integrated calcium signal over a prolonged period of time. This modality of prolonged stimulation by low levels of agonist has been shown to be what is required to achieve cytoprotective effects via 7 receptors [ 227 ] and is probably important for other roles played by 7 receptors in the brain [ 432 ] and in non neuronal tissues [ 126 ] Therefore, since it is likely that in vivo 7 receptors are exposed to low level stimulation (via tonic choline and diffuse phasic release of ACh), receptor activation, based on partial occupancy of the multiple binding sites, may be an important functional modality mediating the cytoprotective and perhaps also the cognitive effects documented for 7 selective agonists.

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122 Figure 5 1. Location of the 7L119 residue and the effect of MTSEA on L119C mutant receptors. A) Location of the L119 residue in a homology model of 7 created by Dr. Nicole Ho renstein. The images were created in Deep View, Swiss PdbViewer [ 433 ] from the crystal structure model of the AChBP (PDB 1I9B; Brejc et al 2001). B) The effect of MTSEA treatment (2 mM for 60 s) on the ACh evoked responses of oocytes expressing the 7 L119C mutation in a cysteine null ( 7 C116S) background. In this experiment, peak current responses to 300 M ACh were reduced 99.4 0.2%, and net charge was reduced by 99.7 0.1% (n = 4). Responses to 3 mM ACh were reduced to a similar extent, 97.9 0.3% and 95.9 2.0% for peak current and net charge, respectively (n = 4).

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123 Figure 5 2. Co Expression o f either L119C or Y188F with wild type 7 subunits at varying ratios. A) The probability for the distribution of mutant subunits based on the injected RNA ratios and the binomial theorem. The assumption that receptors are functionally equivalent irrespective of the subunit positions within th e pentameric structure is made. B) Data traces obtained from oocytes expressing the 7 C116S/L119C MTSEA sensitive mutant and the 7 C116S cysteine null pseudo wild type at the ratios indicated. The series of traces on on the right are the responses to prog ressively greater concentrations of ACh obtained after the MTSEA treatment. C) Average net charge values for oocytes expressing the 7 C116S/L119C MTSEA sensitive mutant and 7 C116S cysteine null pseudo wild type at the ratios indicated following treatment with MTSEA normalized to the 300 M ACh control responses before MTSEA treatment. The data plotted are t he means SEM for at least 5 oocytes at each of the ratios tested. See Table 5 1 for curve fit values.

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124 Figure 5 3. Probing the 7Y188F mutan t receptor with selective and non selective agonists. A) Mutation of the 7 tyrosine 188 to phenylalanine reduces sensitivity to low concentrations of ACh with little impact on sensitivity to the responses to the 7 selective agonist 4OH GTS 21 [ 413 ] The upper traces are representative respo nses of oocytes expressing wild type 7 for which 300 M 4OH GTS 21 evoked responses that are 57 4 % the magnitude of the responses evoked by 300 M ACh, in net charge. In contrast, for oocytes expressing 7 Y 188F, 300 M 4OH GTS 21 evoked net charge responses that are 405 55 % the magnitude of the responses evoked by 300 M ACh. B) Oocytes were injected with RNA for 7 Y188F and wild type 7 at (mutant:wild type) 1:0, 5:1, 3:1, 1:1, and 0:1 ratios and then t ested for their relative responses to 300 M ACh and 300 M 4OH GTS 21. The values are plotted in relation to the fraction of 7Y188F RNA injected a t each ratio and are the means SEM of at least 4 oocytes for every condition.

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125 Figure 5 4. The effect of MTSEA on muscle type receptors with mutations homologous to 7 L119C in muscle # $ and % subunits. Representative responses obtained prior to MTSEA treatments are shown as black lines and responses obtained after MTSEA are gray lines. The schematics to the right of the traces represent the subunit composition and disposition of the ACh binding sites for the different receptor subtypes. The asterisks represent the location of the mutations in the complementary face of the agonist binding site.

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126 Fig ure 5 5. ACh concentration response data for muscle type single subunit mutants before and after MTSEA treatment. Oocytes were stimulated alternately with control applications of 30 M ACh and ACh at increasing test concentrations. The oocytes were then treated with 2 mM MTSEA for 60 s before being tested with the same sequence of ACh applications. All of the data were normalized to the individual oocytes' average responses to the five 30 M ACh applications given prior to the MTSEA treatment. The plot s on the right represent the repeated 30 M ACh responses obtained through the course of the entire experiments, normalized to the average pre MTSEA 30 M ACh responses from each cell. The arrowhead indicates the point at which MTSEA was applied. Th e val ues plotted are the means SEM of 5, 3, and 8 oocytes for 1 1 $# L121C, 1 1 % L119C # 1 1 %# L121C, respectively. Fit parameters are listed in Table 5 2.

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127 Figure 5 6. The effect of MTSEA treatment on peak and NP open responses from receptors expressed in BOSC 23 cells. A) Example traces of outside out patches from each condition. A rapid ( ) 0.7 ms) drug application system was used to apply ACh and MTSEA. B) Summary of the effect of MTSEA treatment (5 mM, 60 s) on peak current and NP open responses to 1 mM ACh shown as the average of post MTSEA measurements relative to the pre MTSEA measurements of each patch. The measurements are normalized to the average peak curent and NP open responses from 8 rundown control patches. Asterisks above the values for 1 1 $ L119C # L121C indicate statistical significance (p< 0.01) when compared with values from either 1 1 $# L121C or wild type. See Table 5 3 for values.

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128 Figure 5 7. Single channel traces and fit burs t duration histograms from wild type 1 1 $# and 1 1 $# L121C receptors before and after MTSEA treatment (5 mM, 60 s) as indicated. Bursts from 1 1 $# L121C receptors before and after MTSEA treatment (indicated or #) are shown on the bottom row in higher time resolution together with the closed duration histo gram from 1 1 $# L121C (before MTSEA treatment) used to define bursts. Currents were sampled at 100 kHz and ultimately low pass filtered to 5 kHz. Each histogram represents the data pooled from at least individual 4 patches recorded under identical condit ions, except for the 10 nM ACh concentration histogram, where data were pooled from 3 patches. Fit parameters are listed in Table 5 4.

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129 Table 5 1. 7L119C ratio experiments net charge data after MTSEA treatment mut:wt ratio I max a EC 50 M 1 :1 1.16 0.05 30 4 3:1 0.83 0.04 31 4 5:1 0.78 0.01 72 3 a Measured relative to AC h maximum before MTSEA treatment Note: Values are the means SEM of at least 5 oocytes Table 5 2. MTSEA effec ts on muscle mutants expressed in Xenopus oocytes Before MTSEA After MTSEA Muscle mutant I max a EC 50 M I max a EC 50 M 1 1 $# L121C (n = 5) 1.30 0.05 12.7 1.5 1.41 0.03 111 8 1 1 % L119C # (n = 3) 2.72 0.08 48.0 4.0 2.40 0.02 126 4 1 1 %# L121C (n = 8) 2.56 0.01 39.1 0.4 1.84 0.04 145 9 1 1 $# (n = 5) 1.15 0.03 4.5 2 0.39 1.35 0.05 3.24 0.41 1 1 %# (n = 7) 1.28 0.003 16.1 0.1 1.53 0.004 22.0 0.11 a Measured relative to average 30 M ACh control before MTSEA treatment Table 5 3. Peak curr ent and NP open measurements from the outside out patch clamp experiments 1 1 $# L121C (control) 1 1 $# L121C 1 1 $ L119 C # L121C 1 1 $# ( n = 8) (n = 12) (n = 9) (n = 8) avg. post/pre NP open a 0.44 (1) 0.28 (0.65) 0.060 (0.14) 0.36 (0.83) SEM 0.13 0.059 0.015 0.085 range 0.078 1.13 0.066 0.69 0.0088 0.11 0.14 0.65 median 0 .38 0.25 0.066 0.36 avg. post/pre peak a 0.41 (1) 0.31 (0.74) 0.030 (0.073) 0.44 (1.06) SEM 0. 081 0.084 0.0027 0.11 range 0.11 0.74 0.051 1 .10 0.020 0.045 0.14 0.78 med ian 0.42 0.23 0.026 0.44 a Normalized values are indicated in parenthesis Note: 1 mM ACh applied before and after 5 m M ACh treatment Table 5 4. Fit time constants from the burst duration histograms condition 1 sem P 1 sem 2 sem P 2 sem 3 sem P 3 sem 1 1 $# ; 1 mM A ch 0.15 0.17 0.24 0.03 12.2 0.04 0.76 0.02 n/a n/a 1 1 $ # after MTSEA 0.18 0.15 0.26 0.03 13.3 0.04 0.7 4 0.02 n/a n/a 1 1 $# L121C; 1 mM ACh 0.13 0.2 0.26 0.04 12.9 0.05 0.74 0.02 n/a n/a 1 1 $# L121C after MT SEA 0 .18 0.08 0.47 0.02 2.02 0.12 0.34 0.03 1 2.6 0.2 0.19 0.03 1 1 $# L12 1C; 10 nM ACh 0.26 0.07 0.43 0.02 4.66 0.04 0.57 0.02 n/a n/a Note: values are indicated in ms and P values indicate fraction of total events from histogram fits

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130 CHAPTER 6 INVESTIGATION OF THE MOLECULAR MECHANISM OF THE ALPHA7 NICOTI NIC ACETYLCHOLINE RECEPT OR POSITIVE ALLOSTER IC MODULATOR PNU 120596 PROVIDES EVIDENCE FO R TWO DISTINCT DESEN SITIZED STATES Introduction Alpha7 nAChRs are considered potentially important therapeutic targets, but the development of selective agonists has been hindered by the concern that endogenous cholinergic function will be disrupted by an agonist based therapeutic approach. Therapeutic strategies utilizing PAMs may allow this is sue to be avoided [ 322 ] The 7 nAChR is a good candidate for allosteric potentiation due to its intrinsically low P open PNU 120596 is one of the most well known 7 PAMs due to its ability to profoundly enhance the amplitude and dramatically prolong agonist evoked responses. In addition, application of PNU 120596 evokes currents 7 receptors that have been desensitized by previous exposure to agonist [ 318 325 ] Here, the potentiation of 7 mediated responses by PNU 120596 was studied via receptors expressed in Xenopus ooc ytes and outside out patches from BOSC23 cells. Two distinct forms of 7 desensitization are identified; one form of desensitization is destabilized by PNU 120596 (D s ) and the other form of desensitization is insensitive (D i ) to reversal by the PAM and is in fact, promoted by strong activation. The onset of D i is so pronounced that large macroscopic currents from outside out patches decay to the point that PNU 120596 potentiated bursts from individual 7 channels are observed, enabling the properties of PNU 120596 potentiated single channel bursts to be measured. The outside out patch clamp experiments demonstrate that PNU 120596 has a remarkable effect on 7 single channel currents, prolonging the duration of an average single channel opening bursts by approximately 100,000 fold.

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131 Results M y contribution to the following results was the patch clamp experiments, which were performed in parallel with the Xenopus oocyte experiments conceived by Dr. Roger Papke. Only the oocyte experiments that are most re levant to the experiments I performed are considered here, for the complete body of published work see [ 376 ] ACh Evoked R esponses of 7 nAChR E xpressed in Xenopus O ocytes The concentration response relationships of heteromeric nAChR macroscopic response s determined from peak current s and net charge are superimposa ble (Figure 6 1A). During a typical application of ACh the response profi le closely matches the rate of agonist applications. The processes of activation and desensitization equilibrate to from a phase of relatively steady current, or a plateau, that decays as agonist is removed [ 213 ] In contrast to heteromeric nAChRs, the concentration response relationships of the homomeric 7 nAChR for peak currents an d net charge are non superimposa ble. This occurs because the responses of 7 nAChR evoked by high concentrations of agonist (Figure 6 1B) reach a peak before solution exchange is complete [ 213 ] The application of high agonist concentrations produce large peak currents, which are indicative of synchronous channel activation, but do no t increase total receptor activation measured by net charge because high agonist concentrations rapidly promote the entry of the receptor into a non conducting desensitized state. A documented effect of the type II 7 PAM PNU 120596 is to reverse or destabilize one or more forms of 7 receptor desensitization. As shown in Figure 6 1C, the concentration response relationships for peak current and net charge evoked by ACh and recorded in the presence of PNU 120596 wer e superimposa ble, as is the case with heteromeric

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132 nAChR. This observation is consistent with the hypothesis that PNU 120596 destabilizes the rapid form of desensitization that is unique to 7. ACh Evoked R esponses of 7 nAChR Expressed in BOSC 2 3 C ells i n the Absence and P resence of PNU 120596 A typical response of an outside out patch expressing 7 to rapid application ( % 0.7 ms) of 60 M ACh is shown in Figure 6 2A. Channel openings occur with highest frequency immediately after application of ACh and t hen quickly disappear despite continued application of agonist. Individual 7 channel openings are extraordinarily brief, and appear primarily as isolated events rather than bursts of openings. At a bandwidth of 10 kHz, the average duration of the appare nt 7 single channel openings evoked by 60 M ACh is 113 7 s (n = 187 openings, 13 patches). Considering that this short duration approaches the temporal resolution limit of 40 s many openings probably occurred that were too short to be reliably dete cted, and the average 7 open duration is over estimated. Figure 6 2B displays a histogram of the apparent channel openings fit by a single exponential function. Assuming the distribution of the missed openings follows the fit exponential function to the ordinate, the corrected average 7 single channel open duration is 59 5 s. At a holding potential of 70 mV, the average single channel amplitude of the apparent 7 openings was 6.22 0.08 pA. Given that individual 7 openings were so short in dura tion, this value may be an under estimate of the true single channel amplitude. A scatter plot of channel amplitude versus open duration showed no clear overall relationship. However, all openings > 300 s in duration had single channel amplitudes betwee n 6.8 pA and 7.6 pA (not shown).

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133 The type II PAM PNU 120596 had a profound impact on responses evoked by 60 M ACh from outside out patches (Figure 6 2C). In the absence of PNU 120596, responses evoked by 12 second applications of 60 M ACh are limited due to the low P open intrinsic to 7 and may lead one to believe the patch contains few channels. A follow up application of 60 M ACh with 10 M PNU 120596 to the same patch revealed that in reality the patch contained a multitude of channels, demonstra ting the massive potential for enhancement of 7 responses by PNU 120596. The net charge response evoked by co application of 60 M ACh and 10 M PNU 120596 was on average 1.35 0.42 x 10 5 fold larger than the net charge response evoked by 60 M ACh alon e (n = 4). The peak current of the response evoked by ACh and PNU 120596 co application, divided by the unitary current amplitude of PNU 120596 potentiated currents, provides a lower limit estimate of the number of channels in a patch (N). The number o f channels in each patch is unknown, but is a parameter of interest since it is required to calculate P open The ACh and PNU 120596 concentration combination that evokes maximal peak responses is important to know in order to obtain a lower limit estimate of N that is as accurate as possible. Several experiments were performed with Xenopus oocytes expressing 7 to test various concentration combinations of ACh and PNU 120596 and found that a range of ACh (10 M 100 M) concentrations with 10 M PNU 1205 96 provided comparable maximal peak currents (data not shown). Based on the detected 7 openings in the absence of PNU 120596 and the lower limit of N provided by peak currents in response to 60 M ACh and 10 M PNU 120596 co application, an upper limit e stimate of 7 P open in response to a 12 second application of

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134 60 M ACh of 7.4 x 10 6 3.0 x 10 6 (n = 4) was calculated. It is important to understand that this estimate is strictly an upper limit estimate since N is unknown and potentially greatly unde restimated. Also, it is equally important to appreciate that this estimate includes the non stationary portion of the 60 M ACh evoked responses, immediately after application of ACh when channel activation occurs with highest probability, and that under true steady state conditions the upper limit 7 P open will be much less. Factors Limiting the Potentiating E ffects of PNU 120596 on 7 Mediated C urrents The time and concentration dependence of PNU 120596 potentiation of wild type human 7 nAChR responses was investigated by making repeated applications of 60 M ACh in the presence of varying bath concentrations of PNU 120596 over the course of one hour. With ACh fixed at 60 M, m aximal potentiation of peak current amplitude was achieved with 10 M PNU 120 596 (Figure 6 3A). Interesting concentration dependent effects were seen on the onset and decline of potentiation with varying concentrations of PNU 120596 (Figure 6 3B). In the presence of 0.3 M PNU 120596, potentiation increased throughout the entire 64 minutes and never reached a plateau ; potentiation was significantly (p < 0.01) larger at 64 minutes than at 12 minutes. In the presence of 1 M PNU 120596, potentiation increased during the first 40 minutes and then began to decrease. Potentiation wa s greater at 40 minutes than at 12 minutes (p < 0.05), but not significantly greater at 64 minutes than at 12 minutes. For all concentrations of PNU 120596 $ 3 M, potentiation reached a peak within 12 minutes and then decayed; potentiation at 64 minutes was significantly less than that at 12 minutes (p < 0.05). These data suggest that increasing concentrations of PNU 120596,

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135 and by inference occupancy of the PAM binding sites, promote forms of desensitization that are insensitive (D i ) to PNU 120596 and t hat accumulate over time. Likewise, the ability of increasing agonist concentrations, and by inference increasing agonist occupancy, to promote D i is demonstrated by first applying 1 M ACh and then applying 100 M ACh to the same outside out patch expressing 7 with a fixed concentration of PNU 120596 (Figure 6 4). Neither the peak current nor the net charge were different between responses evoked by 1 M ACh or 100 M ACh in the pre sence of 10 M PNU 120596 on the time scale of these experiments. However, the onset and decay of the potentiated responses were different. Responses evoked by 1 M ACh in the presence of 10 M PNU 120596 had significantly (p < 0.05) slower 10% 90% rise times than responses evoked by 100 M ACh and 10 M PNU 120596 (12.3 5.1 s versus 1.4 0.5 s; n = 4). Responses evoked by 100 M ACh tended to peak relatively rapidly and then decay despite the continued application of ACh and PNU 120596, while respons es evoked by 1 M ACh generally showed little to no decay on the time scale of these recordings. The ratio of the net charge during the last quarter of the response relative to the first quarter of the response was 1.35 0.28 with 1 M ACh, whereas the r atio was 0.39 0.08 in the 100 M ACh condition. The average 90% 10% decay time of 100 M ACh and 10 M PNU 120596 responses was 26.14 3.52 s (n = 4). In order to test the hypothesis that the induction of D i is dependent on receptor activation, oocy tes were treated with one of two different stimulus protocols (Figure 6 5). In the continued presence of 3 M PNU 120596, oocytes were either repeatedly stimulated with 10 M ACh or with 10 M ACh applications in alternation with stronger

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136 stimulation by 3 00 M ACh. As shown in Figure 6 5B, the 10 M ACh evoked responses of cells stimulated alternately with 300 M ACh reached a level of maximal potentiation relatively quickly and then showed a decline, consistent with the induction of D i In contrast, the 10 M ACh evoked responses of oocytes that did not receive alternating application of 300 M ACh were slower to reach the same level of maximal potentiation, but also no decline in potentiation occurred over the course of an hour. Together these results suggest that both agonist and PNU 120596 concentrations affect the time course of potentiated responses, with faster onset of potentiation and subsequent decay occurring at high concentrations, and low concentrations producing relatively slow rising, but longer lasting currents. Although 7 nAChRs desensitize rapidly in response to high concentrations of agonist, the desensitization is also rapidly reversible once agonist has dissociated from the receptor [ 229 ] As shown in Figure 6 6A, if repeated co applications of 60 M ACh and 10 M PNU 120596 to oocytes were stopped during the period of time when D i was developing and later reinitiate d, the receptors became resensitized to the effects of the PAM. Likewise, recovery from D i occurred in outside out patches in experiments performed on a much shorter timescale (Figure 6 6B), suggesting that PNU 120596 insensitive forms of desensitization are reversible. Single Channel B ursts of 7 nAChR P romoted by PNU 120596 Because the potentiation of responses by PNU 120596 is so profound, the probability of observing single channel currents potentiated by PNU 120596 was initially hypothesized to be low. However, the decay of current was so pronounced with prolonged long applications of 300 M ACh and 10 M PNU 120596 that single channel

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137 bursts potentiated by PNU 120596 were revealed once the majority of channels entered the D i state(s) (Figure 6 7A). From three patches with sections of cont inuous data > 60 seconds lacking simultaneous channel openings, the steady state NP open in 300 M ACh and 10 M PNU 120596 was 0.40 0.04. Given the length of the single channel bursts and the lack of simultaneous openings in these traces, it seems that the number of activatible channels at any time is greatly limited by D i It is unknown whether the openings in these traces all arise from one resilient channel that escaped and remained resistant to D i or whether the openings arose from multiple channel s as they temporarily escaped D i There appeared to be two qualitatively distinct groups of single channel openings in the presence of PNU 120596 (Figure 6 7A). One class of PNU 120596 potentiated opening appeared as relatively brief events that primari ly occurred in isolation, and the other, more common, type of PNU 120596 potentiated openings appeared to occur as long bursts of openings separated by very short closures to either fully closed or subconductance states (see below). Most of the brief open ings observed in the presence of PNU 120596 were <1 m in duration, occasionally lasting approximately 8 ms (Table 6 1). A major component of the isolated openings evoked in the presence of PNU 120596 was fit with a time constant of 120 6 s, which is similar to the average apparent open duration in the absence of PNU 120596. On average, the protracted bursts of openings potentiated by 10 M PNU 120596 persisted for 5.48 0.40 s (n = 217, 26 patches). The majority of the apparent intrab urst closures were approximately 100 s 200 s; however, occasionally intraburst closures longer than approximately 10 ms were observed. The subconductance events

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138 were approximately 400 s or less in duration and were interrupted by many fast full channel closures (Figure 6 7A inset, Table 6 1). There appeared to be two distinct classes of the long PNU 120596 potentiated bursts; the distinction was made based on the duration of the intraburst openings (Figure 6 7B, Table 6 1). Approximately 27% of the bu rsts analyzed exclusively contained intraburst openings of approximately 5 ms in duration (class I bursts) and 73% of the bursts exclusively contained intraburst openings of approximately 30 ms in duration (class II bursts); no clear relationship was appar ent between burst length and average intraburst open durations (Figure 6 7B). The apparent closed and subconductance duration intervals were similar between the two classes of bursts (Table 6 1). The reason for the two types of bursts is unclear, but may be related to occupancy of ACh and/or PNU 120596 at their respective binding sites during the burst of openings. At a holding potential of 70 mV, the single channel amplitude of openings potentiated by PNU 120596 was 7.76 0.08 pA, and the subconduct ance level amplitude was 3.59 0.02 pA. The current voltage relationship of currents evoked in the presence of PNU 120596 showed strong inward rectification, a reversal potential of 6.93 1.01 mV, and a single channel chord conductance of 129 3 pS t hrough the linear section of the relationship (n = 6, data not shown). Given that PNU 120596 appears to have little, if any, effect on channel conductance [ 318 ] the conductance measured in the presence of PNU 120596 is likely to be a reasonable estimate of single channel 7 conductance in the absence of PNU 120596. This measurement of 129 3 pS is higher than the previously published values of 60 90 pS [ 215 216 ] but

PAGE 139

139 the difference is probably related to the limited ability to accurately measure 7 open channel a mplitude, since non potentiated 7 openings are so brief. A phenomenon observed with currents from the outside out patches that have been activated with ACh and potentiated by PNU 120596 was a postponed return to baseline after the removal of external ACh (Figure 6 8A). For single channel bursts activated in the presence of 300 M ACh and 10 M PNU 120596, currents persisted for 2.57 0.2 s following the removal of external ACh (n = 143, 15 patches). There was no detectable change of intraburst open dura tions, nor was there an apparent trend toward change in intraburst open duration over time after ACh removal. The protracted currents were not an artifact of slow or incomplete solution exchange based on three arguments. First, following data collection each patch was blown off the tip of the pipette and solution exchange profiles were determined by measuring changes in holding current upon moving diluted saline over the open pipette tip with the piezoelectric stepper (Figure 6 8B). Data were analyzed on ly if the solution exchange profile measured in this way was clean and occurred rapidly, within ~700 s (10% 90% rise time). Typical solution exchange times measured with this method were between 400 s and 700 s. Second, there was a sharp reduction in current noise that was intimately associated with the removal of external ACh (Figure 6 8B). This reduction in noise was likely due to relief of channel block by ACh, given that a relatively high concentration was used as the stimulating agonist [ 412 ] This reduction in current noise was highly significant (p <3 x 10 15 ), as measured by changes in the standard deviation from the mean open current before and after removal of external ACh ( = 0.44 0.008 versus 0.31 0.003). Third, replacement of external ACh with the competitive 7

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140 antagonist MLA greatly attenuated the duration of the protracted currents (see below). The currents that persisted long after the removal of external ACh we re consistent with prolonged retention of ACh at the binding sites when PNU 120596 is applied. However, this observation could also be explained by maintenance of the channel in a reverberating conducting condition by PNU 120596 after dissociation of ACh on termination of external ACh application. An attempt to test the hypothesis that PNU 120596 prolongs the retention of ACh to its binding site was made by removing external ACh and replacing it with 3 M MLA. The initial reasoning behind this experiment was if the affinity of the agonist binding site for ACh were increased by PNU 120596, application of the high MLA concentration would have little effect on the duration of protracted currents given that the channel was both occupied by ACh and in an activated condition on application of MLA. However, when ACh was replaced with MLA, the currents persisted for 0.22 0.03 s (n = 25, 3 patches) after removal of external ACh, a significant (p <1 x 10 5 ) red uction of the protracted current duration (Figure 6 8C). It is noteworthy that the effect of MLA, which is normally considered a competitive antagonist, had the appearance of an inverse agonist in these experiments, given that channels were active when ML A was applied, and MLA shortened burst activity under conditions when no external ACh was available for competition. Prior to moving the patches out of the solution containing 300 M ACh, it is reasonable to assume that the five agonist binding sites of 7 were occupied by ACh at high probability. Further, it seems likely that after the removal of external ACh, agonist molecules began to dissociate one by one until the last ACh molecule dissociated, perhaps resulting in termination of the burst. When ext ernal ACh was

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141 removed, and individual ACh molecules began to dissociate, MLA may have had the opportunity to bind the receptor in the vacant binding sites and terminate the burst before all ACh molecules had dissociated. The data suggest that binding of o nly one or two molecules of MLA may be sufficient to inactivate the entire receptor and that MLA may actively inhibit channel gating, perhaps by stabilizing non conducting receptor conformations, rather than simply occupying the cavity where ACh binds to p roduce channel activation. Discussion The initial characterizations of the type II PAMs PNU 120596, TQS and A 867744 reported that these agents reverse or eliminate desensitization [ 434 ] This finding was supported by observations that potentiated currents did not substantially decay on the time scale of the experiments and th at previously desensitized receptors could be de desensitized with application of a type II PAM [ 318 325 326 ] The results presented here show that at least two distinct forms of 7 desensitization exist; these are distinguished by their stability in the presence of PNU 120596. The published literature lacks a formal description of D i but there are some published observations from other research groups that are interpreted as con sistent with the existence of PAM insensitive D i states. First, w hen agonist concentrations are increased in the presence of a fixed PAM concentration, responses are generally enhanced over the full agonist concentration ranges, but the magnitude of poten tiation often tends to peak at intermediate concentrations and actually decrease at the higher agonist concentrations [ 318 321 325 ] A similar phenomenon is sometimes observed when modulator concentrations are increased in the presence of a constant agonist concentration [ 320

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142 321 ] Second, applications of TQS [ 325 ] or A 867744 [ 326 ] were shown to re activate receptors that had been desensitized by a high concentration of agonist and produced a non decaying curre nt on the time scale of the experiments, consistent with reversal of D s states to conducting states. However, the peak of the potentiated current in the presence of either TQS or A 867744 was only ~50% of the peak response recorded in the absence of PAM, indicating that only a fraction of the receptor populations were actually conducting current at any given moment. Third, several applications of 30 M nicotine, with short inter stimulus intervals, in the continued presence of PNU 120596 produced successively smaller responses [ 316 ] indicative of the accumulat ion of modulator insensitive D i states. Fourth, an bungarotoxin sensitive and PNU 120596 dependent increase in calcium response from bovine chromaffin cells was only observed when low (1 M) concentrations of nicotine were used to stimulate the cells. At concentrations of $ 3 M nicotine, neither PNU 120596 nor bungarotoxin had an effect on the response, indicating that the 7 component was lost by the higher nicotine concentrations [ 435 ] In addition, when the 7 component of the total calcium response was isolated, increased concentrations of the 7 agonist PNU 282987 with a fixed PNU 120596 concentration, resulted in decreased 7 dependent calcium responses [ 435 ] The strong apparent induction of D i in the outside out patches, yet extremely long bursting activity of individual channels, suggests PNU 120596 is capable of having very large effects on just a few channels at a time. While the net charge of an oocyte whole cell current is increased as much as 500 fold by PNU 120596, the net charge of a single channel burst for a PNU 120596 primed channel is approximately 100,000 times

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143 greater than th at of a non potentiated 7 single channel opening. These two numbers are resolved if only 1 in 200 receptors are active in the bursting states at any moment during a PNU 120596 treatment. While the data suggest that high levels of agonist occupancy gener ally disfavor channel opening through stabilization of D i the mode by which a few resilient PNU 120596 primed channels escape D i and enter the hyper bursting state, despite high agonist and PAM occupancy, is less clear. These observations indicate that PNU 120596 primed currents should be used with caution for setting a lower limit on N, meaning the 7 P open estimate following application of 60 M ACh could be much less than 7.4 x 10 6 Even if this upper limit is greatly overestimated, the data in Figure 6 2 argue strongly against the assumption that 7 P open is high immediately following rapid expo sure to agonist, as previous ly proposed [ 436 ] The observation that bursting activity of single channels in the presence of PNU 120596 persist for approximately 2.5 seconds after removal of external ACh is consistent with the prolonged retention of ACh once channels enter the PNU 120596 primed bursting mode. This observation, in combination with the finding that the onset of potentiation by PNU 120596 is depende nt on channel activity, suggests the possibility that agonist and PAM binding sites mutually interact. This possibility is supported by a recent report that the potency of the agonist PNU 282987 to evoke calcium responses in IMR 32 cells was increased wit h higher PNU 120596 concentrations, and, vice versa, the potency of PNU 120596 to potentiate responses was increased with higher PNU 282987 concentrations [ 437 ] In addition, computer docking simulations predicted that binding of PNU 120596 to the proposed

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144 transmembrane cavity has higher aff inity in an "open" channel model than in a "closed" channel model [ 344 ] Other experiments presented in Williams et al ., 2011 that were designed by Dr. Papke and performed by other members of the laboratory further support the existence of D i states [ 376 ] The peaks of potentiated currents were shown to occur faster with stronger stimulation. However, the height of the peak currents and sustained equilibrium currents over time were smallest when higher concentrations of agonist and PNU 120596 were applied, consistent with the induction of D i states under conditions o f strong stimulation. In addition, the competitive antagonists MLA and dihydro erythroidine were shown to be able to modulate the equilibrium between D s and D i states. Notably, competitive antagonists appeared to actually have the effect of increasing currents evoked by choline and PNU 120596 in a manner that was dependent on the agonist and antagonist concentrations. Furthermore, benzylidene anabaseine agonists of 7 were shown to stabilize PNU 120596 sensitive states to varying degrees, despite struc tural similarity, agonists can preferentially stabilize D s or D i states. The ability of agonists to preferentially stabilize receptor states may be important in the rational design of 7 agonists if one type of state mediates a desired therapeutic effect. Interestingly, 7 mediated signal transduction has been observed under conditions when ion channel activation is not apparent [ 438 ] leaving the intriguing possibility open that 7 desensitized states are not inactive, but functionally meaningful. Activation of 7 can have either protective or toxic effects depending on the mo de of stimulation [ 227 ] and the potential fo r enormous potentiation of 7 responses by type

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145 II PAMs invites the concern that potentiation of 7 with type II PAMs may bring receptor activation to dan gerously high levels [ 434 ] There are contradicting reports in the published literature regarding the in vitro cytotoxicity profile of PNU 120596; in the two cases where PNU 120596 w as found to be toxic [ 316 321 ] the stimulating agonist was the weakly potent 7 agonist choline at a concentration of ~100 M, which is well below the published EC 50 value [ 214 ] and potentially falls wi thin the window of strong 7 potentiation where D i is avoided. In contrast, PNU 120596 was reported to lack a significant cytotoxicity profile when co applied with 10 M of the potent 7 agonist PNU 282987 [ 374 ] Given that the published EC 50 value of PNU 282987 in the presence of 3 M PNU 120596 is only 200 nM [ 325 ] and D i induced by high PNU 282987 concentrations is apparent in bovine chromaffin cells [ 435 ] the existence of D i states may provide an intrinsic safety mechanism and might account for the discrepancy in the published literature regarding type II PAM toxicity profiles.

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146 Figure 6 1. The basic characterization of 7 macroscop ic currents, comparison to currents from heteromeric 4 2 nAChR, and the effects of PNU 120596. The data in this figure were collected by OpusXpress technicians and were analyzed by Dr. Roger Papke. A) ACh concentration response relationships for peak cu rrents and net charge from Xenopus oocytes expressing human ( 4) 3 ( 2) 2 nAChR. B) ACh concentration response relationships for peak currents and net charge from Xenopus oocytes expressing human 7 nAChR. C) ACh concentration response relationships in the presence of 10 M PNU 120596 for peak currents and net charge from Xenopus oocytes expressing human 7 nAChR. After obtaining two initial control responses to the application of 60 M ACh alone, the bath perfusion system was switched to a solution contain ing 10 M PNU 120596. ACh and PNU 120596 co applications were made with the pipette delivery system. Net charge responses were calculated for a period of 120 s following drug applications. Each data point is the average of at least 4 cells SE M

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147 F igure 6 2. The low intrinsic P open of 7 is enhanced by PNU 120596. Data were obtained from outside out patches pulled from BOSC23 cells transiently expressing human 7 and ric3. A) A typical response evoked by rapid ( ) 0.7 ms) application of 60 M ACh showing the short lived openings that ar e characteristic of 7 Notice that the frequency of cha nnel openings quickly diminished following exposure of the patch to ACh. B) A histogram of the apparent single channel 7 open durations evoked by 60 M ACh (n = 187 open events from 13 patches; 10 kHz bandwidth). Putative openings that could not be clearly distinguished from brief, random noise spikes were excluded from the histogram. The histogram is described by a single exponential function, which predicts that 7 single channel openings are, o n average, approximately 60 s in duration assuming the distribution of openings too short to be detected (<40 s) reasonably follows the fit exponential function. C) Responses produced by 60 M ACh and relative increases produced with co application of wi th 60 M ACh and 10 M PNU 120596. Drug applications and inter stimulus intervals were 12 s and 30 s in duration, respectively.

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148 Figure 6 3. Factors defining and limiting the potentiating effects of PNU 120596 on 7 nAChR expressed in Xenopus oocytes The data in this figure were collected by Opu sXpress technicians and were analyzed by Dr. Roger Papke. A) Maximum potentiation of 60 M ACh evoked responses obtained with different concentrations of bath applied PNU 120596 applied repeatedly at 4 minut e intervals for over 60 minutes. B) The time course of the potentiation of 60 M ACh evoked responses by varying concentrations of bath applied PNU 120596. Note that although each point represents the average of at least four oocytes, error bars have bee n omitted for clear presentation of the kinetic differences.

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149 Fig ure 6 4. Agonist concentration dependence on the onset and decline of potentiation by PNU 120596 in outside out patches containing 7 receptors. Comparison of responses from the same pa tch potentiated by 10 M PNU 120596 and evoked by 1 or 100 M ACh. Drug applications and interstimulus intervals were 45 and 55 s in duration, respectively. The ensemble average of all four current traces was created by first normalizing each data point of an individual trace to the peak current of that trace. Then, the average value of the normalized currents was calculated for each data point.

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150 Figure 6 5. Activity dependence of PNU 120596 potentiation onset and decline. The data in this figu re were collected by OpusXpress technicians and analyzed by Dr. Roger Papke. A) Representative data traces showing the repeated stimulation of 7 expressing oocytes in the presence of 3 M PNU 120596. Cells were either stimulated repeatedly with 10 M ACh (upper trace), or 10 M ACh alternating with applications of 300 M ACh (l ower trace). B) Average data SEM for 10 M ACh evoked responses obtained with the two protocols illustrated in panel A. Note that maximal potentiation was achieved more rapidly when there were alternating applications of the high concentration of ACh; however, potentiation subsequently declined.

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151 Figure 6 6. R ecovery from PNU 120596 in sensitive desensitization. A) These data were collected by OpusXpress technicians and were analyzed by Dr. Roger Papke. Xenopus oocytes expressing human 7 were stimulated repeatedly with 60 M ACh in the presence of 10 M PNU 120596. To determine if the decline in ACh evoked responses represented a reversible form of PNU 120596 desensitization, some cells (solid points) received 6 applications of PNU 120 596 solution alone rather than repeated co applications of ACh plus PNU 120596. Subsequent application of ACh plus PNU 120596 to the cells that had received a respite from the repeated ACh stimulations returned to a level that was not different the previo usly maximal level of potentiation, and significantly greater than (p < 0.01) that of the cell receiving continuous stimulation. B) Responses potentiated by 10 M PNU 120596 and evoked by 300 M ACh show a rapid rise and subsequent decay despite the continued presence of ACh and PNU 120596, indicative of stabilized D i states. Recovery from D i induced by co application of 300 M ACh and 10 M PNU 120596 occurs ra pidly. Identical 45 s drug applications were separated by 5 s inter stimulus intervals.

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152 Figure 6 7. Single channel 7 bursts evoked by 300 M ACh and potentiated by 10 M PNU 120596. Data were obtained from outside out patches pulled from BOSC23 cell s transiently expressing human 7 and ric 3. A) Continuous data demonstrating the bursting characteristics and high steady state P open of single channel openings potentiated by PNU 120596. B) Fit histograms displaying apparent open durations from 7 chann els activated by 300 M ACh and potentiated by 10 M PNU 120596. Shown are histograms compiled from currents potentiated by PNU 120596 that occur as brief events in isolation, and in long groups of openings as bursts. Two classes of bursts are distinguis hed based on the average intraburst open duration, which is demonstrated below with a scatter plot of average intraburst duration versus total burst length.

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153 Figure 6 8. Single channel 7 bursts evoked by 300 M ACh and potentiated by 10 M PNU 120596 persist despite the removal of ACh and in a MLA sensitive manner. Data were obtained from outside out patches pulled from BOSC23 cells transiently expressing human 7 and ric3. A) PNU 120596 potentiated channel openings persist for seconds after removal of external ACh. Left, an example of a protracted macroscopic current that persisted for approximately 8.3 seconds after external ACh removal. Right, single channel bursts showing protracted currents of various durations after external ACh removal. B) T he protracted currents were not an artifact of solution exchange. Above, solution exchanges typically occurred within 400 s 700 s, as measured by open tip recordings performed following data collection from each patch. Below, a sharp reduction in current noise was associated with the removal of external ACh. The noise was likely due to channel block by agonist, which di sappeared as external agonist was removed. C) Application of the competitive antagonist MLA following removal of external ACh shortened the duration of protracted currents. All currents were obtained from the same patch.

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154 Table 6 1. Fit time constants f rom event duration histograms in the presence of 300 M ACh and 10 M PNU 120596 Isolated openings Class I bursts Class II bursts Type of event P sem sem P sem sem P sem sem Intraburst closures n/a n/a 0.91 0 .06 0.07 0.10 0.77 0.14 0.16 0.18 n/a n/a 0.07 0.04 0.51 0.82 0.21 0.15 0.57 0.6 n/a n/a 0 .02 0.03 9.16 4.52 0.02 0.04 13.2 2.5 Intraburst subcond n/a n/a 0.80 0.12 0.08 0.09 0.82 0.07 0.14 0.09 n/a n/a 0.20 0.04 0.40 0.20 0.18 0.09 0.37 0.24 Intraburst openings 0.54 0.02 0.12 0.06 n/a n/a n/a n/a 0.30 0.02 0.71 0.11 n/a n/a n/a n/a 0.16 0.02 8.23 0.1 4 1 5.35 0.02 1 27.25 0.02 Note: values are indicated in ms and P values indicate fraction of total events from fit H istograms

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155 CHAPTER 7 A NOVEL CELL LINE ST ABLY EXPRESSING HUMA N ALPHA7 NICOTINIC ACETYLCHOLINE RECEPT ORS REVEALS THAT PNU 120596 POTENTIATION AND CYTOXICITY ARE ATT ENUATED AT PHYSIOLOG ICAL TEMPERATURES Introduction Within the last 20 years both academic and industrial labs have discovered numerous agonists with selectively for 7 [ 314 ] These efforts have been justified by large amounts of pr e clinical in vitro and in vivo behavioral data from several independent laboratories suggesting that activation of 7 may provide cytoprotection, enhances performance in a variety of behavioral tasks thought to be related to cognitive function, and reduce s auditory gating defici ts modeled after those seen in s chizophrenia [ 294 295 ] An alternative therapeu tic approach based on allosteric modulation has gained momentum in recent years with the discovery many structurally diverse PAMs of 7 nAChR [ 322 ] In cases where activation of 7 is necessary for a desired effect [ 291 ] a PAM based therapeutic approach offers several potentia l advantages over agonist based strategies. For example, PAMs may be able to offer increased selective targeting of a desired receptor subtype since PAM binding sites presumably have less evolutionary pressure than agonist binding sites which must accomm odate the endogenous ligand. In addition, the temporal firing dynamics of native cholinergic signaling may be better conserved since PAMs would theoretically only augment the response provided by the natural release of ACh However, a PAM based strategy is also subject to some important limitations that need to be appreciated. First, PAMs will require sufficient endogenous neurotransmitter release, which could be an issue in neurodegenerative disorders. Second, desensitized states have been identified t hat are stable in the

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156 presence of PNU 120596 (see chapter 6 and [ 376 ] ) This type of desensitization (D i ) appears to be promoted by strong channel activation and high fractional occupancy of both agonist and modulator binding sites. Although the implications of such states have not been addressed in vivo, it is conceivable that D i states may accumulate over time with the prolonged presentation of a PAM. Thir d, toxicity poses a real threat due to excess activation of the calcium permeable pore. Indeed, strong activation of 7 channels has been previously shown to be cytotoxic [ 371 373 ] The issue of PAM induced toxicity in vitro has been evaluated by three independent groups and while the data agree that type I PAMs appear to lack in vitro cytotoxicity, th e data are contradicting regarding the toxicity of the type II PAM PNU 120596. Fourth, the ability of 7 PAMs to potentiate 7 mediated responses may be significantly reduced at p hysiological temperature [ 439 ] This has obvious implications for understanding basic science questions about how these compounds work and, more importantly, for their development as human therapeutics. These results also raise questions regarding the relevance of in vitro electrophysiological experiments performed at non physiological temperatures, which are preferentially performed due to technical reasons including the fact that pa tches are less stable at higher temperatures and the requirement for additional equipment in order to precisely control the temperature of experimental solutions. Here, a novel HEK293 cell line stably expressing the human 7 nAChR was created and used to characterize the in vitro temperature dependence of and cytotoxicity profile of PNU 120596. The findings suggest that the potentiating activity of PNU 120596 is reduced at physiological temperature, but that some endogenou s

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157 factor s such as serum albumins may partially restore the ability of PNU 120596 to potentiate responses at 37 ¡ C. Results Expression of hr ic3 and h 7 mRNA in Hygromycin and G418 Resistant C lones Total mRNA was isolated from each hygromycin and G418 re sistant clone a nd tested for the presence of hr ic3 and h 7 mRNA through RT PCR. As expected, untransfec ted HEK293 was negative for hr ic3 and for h 7, but bands cor responding to the expected nucleotide length were observed for both hr ic3 and h 7 from the a ntibiotic resistant cell lines (Figure 7 1A). Messenger RNA for GAPDH, a common housekeeping gene, was probed as a positive control to verify that the RT PCR protocol wa s successful in the case that hr ic3 and h 7 bands were absent. Identification of the h 7 Protein via Western Blot in Antibiotic Resistant C lones Immunoprecipitation and w estern blots for 7 protein from whole cell lysates were performed by Clare Stokes and Monica Santisteban. As expected, no labeling was observed from untransfected cells an d cells stably expressing h ric3. In contrast, some labe ling was observed f rom cells transfected with h 7 while stronger labeling was seen in ce lls stably expressing both h 7 and h ric3 (Figure 7 1B). The 7 protein detected in this Western Blot is an a ggregation with a molecular weight > 220 kDa; t he expected molecular weight of an 7 pentamer is 280 kDa. Labeling of HEK h 7/hric3 C ell s with Alexa488 C onjugated B ungarotoxin Intact cells were labeled with Alexa488 conjugated bungarotoxin to qualit atively verify surface expression of 7 nAChR and to illustrate the distribution of receptor expression in this cell line. As expected, the untransfected HEK293, HEK h 7, and

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158 HEK hric3 cell lines were not labeled with the fluorescent tox in. In contrast, the HEK h 7/hric3 cell line was labeled by the fluorescent bungarotoxin, and in a competitive manner with 1 mM nicotine (Figure 7 2). The labeling appears in non continuous clusters, suggesting that surface expression of 7 in this cell line may be non uniform. Similar patterns of labeling by fluorescent dye conjugated ligands of 7 have been seen in other cell lin es and in cultured neurons [ 440 441 ] Specific Binding of [ 125 I] Bungarotoxin to I ntact HEK h 7/hric3 C ells Specific labeling with 3 nM [ 125 I] bungarotoxin was observed in intact HEK h 7 /hric3 cells while no specific labeling was detected in u ntransfected HEK293 cells treated in parallel (Figure 7 3 A) From three saturation binding experiments to intact cells the K d of bungarotoxin is 991 67 pM and the B max is 7.71 x 10 8 1.28 x 10 8 pmol/cell (Figure 7 3B) This B max translates to an average of 9,284 1,544 receptors expressed/cell assuming that 5 molecules of radiolabel ed bungarotoxin bind each 7 receptor. From 34 HEK h 7/hric3 cells, the average peak current and net charge (during one second) evoked by 300 M ACh was 163 26 pA and 9,775 1,458 pA x ms, respectively. The single channel amplitude of 7 channels (potentiated by PNU 120596) was determined to be approximat ely 7.8 pA [ 37 6 ] ; t h is means that ~21 7 channels were open at the peak of an average current If an average cell expresses 9,300 7 ion channels and all of those ion channels are equally activatible just prior to the agonist stimulation, the maximal 7 P open at the peak of the current is approximately 0.0023 Assuming an average single channel open life time of 0.1 ms [ 376 ] an average net charge response to a one secon d application of 300 M ACh contains ~12,532 channel openings. Based on these numbers, the average number of times an individual

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159 channel opened during the one second of 300 M ACh application is 1.35. These numbers are certainly rough estimates, but they suggest that t he ins tantaneous P open of 7 is never high not even immediately after the presentation of a strong agonist stimulus and that an individual 7 channel probably opens 3 times or less before becoming desensitized in response to 300 M ACh Under steady state con ditions where ACh is presented for prolonged periods of time, the 7 P open will be substantially less than the estimated maximum of 0.0023 due to desensitization. The peak current of PNU 120596 potentiated responses from outside out patches was previously used to obtain a lower limit of the number of channels (N) in the patch. This lower limit of N was used to estimate that the upper limit of the 7 P open during a 12 second application of 60 M ACh is 7.4 x 10 6 3 .0 x 10 6 (see Chapter 6). ACh Evoked R esponses from HEK h 7/hric3 Cells and I n hibition of those Currents with MLA in a Concentration Dependent M anner ACh evoked whole cell curren ts were recorded from HEK h 7/hric3 cells with patch clamp electrophysiology. The ACh concentration response relat ionship resulted in non superimposable curves for peak current and net charge measurements, as expected for 7 receptors (Figure 7 4A and B). The EC 50 values determined from peak currents and net charge were 167 20 M and 26 6 M, respectively These values are very similar to those previously reported for human 7 nAChRs expressed in Xenopus ooctyes [ 214 ] The curve for net charge was fit between the 1 M ACh and 300 M ACh points since the net charge was reduced from the maximum at ACh concentrations above 300 M. A unique feature of the 7 receptor is the concentration dependent desensitization that rapidly occurs with applications of high agonist

PAGE 160

160 con centrations. In fact, this form of desensitization occurs more rapidly than solution exch anges can be practically made [ 213 ] The reduction s in net charge at high concentrations seen in this experiment were more pronounced than normally occurs in oocyte experiments. This was likely due to differences in the presentation of agonist; drug applications were made wi th a system that provided solution exchanges on the order of several milliseconds versus the drug delivery that occurs on a time scale of 3 4 seconds in a typical oocyte experiment T he rapid application of high agonist concentrations produced synchronous activation and desensitization of the 7 receptor population, resulting in extremely sharp macroscopic responses with minimal area ( Figure 7 4B) This is also demonstrated by comparing the rise times and rise slopes with increasing concentrations of ACh ; the 10 90% rise times became shorter a nd the 10 90% rise slopes became steeper as ACh concentrations increased (Table 7 1). The ACh evoked responses recorded from HEK h 7/hric3 cells were sensitive to inhibition by the 7 selective antagonist MLA in a concentration dependent manner (Figure 7 4C and D). In this experiment, the ACh concentration was fixed at 170 M (the EC 50 for peak currents determined above), with increasing co applications of MLA. No preincubation with MLA was made in these experiments. The IC 50 of MLA measured in this paradigm was 2.70.4 M. At first glance this may seem like a rather high value for MLA, but when one considers that IC 50 values are dependent on variables such as agonist concentration, timing, and duration of antagonist applications, this value is reas onable. For example, high affinity inhibition of 7 mediated responses is obtained only when MLA is pre incubated prior to the application of agonist [ 133 442 ] In addition, the IC 50 value of MLA in an oocyte experiment utilizing a similar paradigm to

PAGE 161

161 the one used here (no MLA pre incubation) was determined to be 1.20.2 M with an ACh concentration of 60 M [ 443 ] Given that the ACh concentration used here was 170 M, or roughly 3 fold higher, the IC 50 of 2.70.4 M for MLA inhibition of responses evoked by 170 M ACh is consistent with previously published data. In Vitro Cytotoxicity Pro file of PNU 120596 in HEK h 7/hric3 C ells The in vitro cytotoxocity profile of PNU 120596 was evaluated in the HEK h 7/hric3 cell line. In contrast to previous studies which tested limited agonist and/or PAM concentrations, the toxicity profile of PNU 120596 was assessed over a range of agonist (choline) and PNU 120596 concentr ations. Based on the recent finding that high concentrations of agonist and PNU 120596 promote non conducting desensitized states that are insensitive to reversal by PNU 120596 [ 376 ] maximal cytotoxicity was hypothesized to occur upon treatment with relatively low concentrations of both agonist and PNU 120596 since this condition produces the greatest degree of ion channel activation over time In addition, the existence of D i states were hypothesized to account for the discrepancy in the literature regarding the toxicity of PNU 120596, given that the two studies reporting PNU 120596 toxicity used 100 M choline as the agonist while the one study that reporte d a lack of PNU 120596 toxicity used a very strong agonist stimulus that potentially stabilized D i states (explained in Chapter 6 ). The hypothesis that temperature might account for the discrepancy is unlikely given that all of the previously published st udies were performed at 37C. Nonetheless, there was great interest in determining if the apparent temperature dependence of PNU 120596 from electrophysiology experiments might translate into measurable differences in cytotoxic effects when PNU 120596 tre atments were incubated at different

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162 temperatures. In these exper iments choline, rather than ACh was used as the stimulating agonist to avoid issues that may accompany th e labile nature of ACh Choline has been shown to selectively activate 7 nAChRs wit h similar efficacy to ACh, although with approximately 1 0 fold lower potency [ 444 ] Initial cytotoxici ty experiments were performed in full rich DMEM media con taining 10% FBS with treatments incubated at 37C. A range of choline concentrations between 0 and 3 mM were co applied with 10 M PNU 120596 and in these experiments cell viabilities were reduced to approximately 15 25% of the controls whenever PNU 120596 was applied (data not shown). Two observations were immediately obvious from the initial experiments. First, PNU 120596 produ ced a great deal of toxicity even at 37C despite the electrophysiological evidence that PNU 120596 potentiates poorly at this temperature (this is addressed below). This finding was in agreement with Dinklo et al 2011 and Ng et al 2007 Second, PNU 120596 applied alone without agonist caused the same degree of toxicity as when applied with agonist. Evaluation of the ingredients in DMEM revealed that the medium contains approximately 30 M choline. In addition, FBS is undefined, and very likely contains an unknown (but probably relatively low) concentration of choline. Experiments were performed to evaluate the onset of toxicity produced by the choline + PNU 120596 treatments, which was determined to occur to a full extent with less than 2 hours of treatment (data not shown). Due to the short incubation time required to produce the toxic effect, it was reasoned that the experiments could be performed in HBSS, a simple buffered solution containin g glucose, without FBS supplementation. When experiments

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163 were performed in HBSS, treatments with PNU 120596 alone failed to produce cytotoxicity and all future experiments were performed in HBSS. Significant toxicity was observed when treatments were incubated at 28 ¡ C in HBSS in a manner that was dependent on the concentration of PNU 120596, and to a lesser extent the choline concentration (Figure 7 5A). In contrast, when choline and PNU 120596 treatments were incubated at 37 ¡ C in HBSS, no signif icant degree of toxicity was observed for any of the choline/PNU 120596 combinations (Figure 7 5B). As a control, all treatments were made in parallel to untransfected HEK293 cells and no treatment of choline and/or PNU 120596 at 28 ¡ C or 37 ¡ C produced sig nificant toxicity (data not shown). Applications of choline alone or PNU 120596 alone did not reduce cell viabilities. No significant toxicity was produced with 1 M PNU 120596 over the range of choline concentrations tested. At 3 M PNU 120596 statisti cally significant toxicity was observed with 1 mM and 3 mM choline co applications. The greatest degree of toxicity was observed when choline was applied with 10 M PNU 120596, with all concentrations of choline tested producing approximately 50% reductio ns in cell viability relative to the controls. With 30 M PNU 120596, significant reductions in cell viability were observed when co applications were made with 100 M and 1 mM choline, but the magnitude of toxicity in this case was less than was observed when treatments included 10 M PNU 120596. Overall, there was no clear relationship between agonist concentration and degree of toxicity as predicted a priori. However, the observation that the magnitude of toxicity is actually decreased with 30 M PNU 120596 treatments relative to 10 M PNU 120596 treatments is consistent with the induction of D i states when modulator concentrations are high, and by inference high

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164 occupancy of PNU 120596 binding sites. In addition, the temperature dependent cytotoxicit y effects of PNU 120596 correlate well to the temperature dependent potentiating activity of PNU 120596 observed in the whole cell elec trophysiology experiments of Sitzia et al 2011 and Dr. Can Peng (see below). To confirm whether the observed toxicity o ccurred via 7 nAChRs, the competitive antagonist MLA was given at varying time points with 100 M choline + 10 M PNU 120596 treatments and incubated at 28 ¡ C. As seen in Figure 7 6, neither a 10 minute pre application n or co application of 10 nM MLA were able to blo ck the choline + PNU 120596 toxicity. However, this is consistent with the recent observation that 10 nM applications of MLA on top of steady state currents elicited by choline + 3 M PNU 120596 actually transiently increase current rather than inhibit cu rrent, suggesting that low concentrations of MLA may alter the equilibrium between D s and D i towards D s [ 376 ] Application of a 10 fold higher MLA concentrat ion either 10 minutes before or with 100 M choline + 10 M PNU 120596 was able to completely block the toxic effect, suggesting the toxicity is mediated by 7 receptors. In contrast, 100 nM MLA was unable to block the effect if it was applied 10 minutes or more after the toxic choline + 10 PNU 120596 treatment. This suggests that the onset of PNU 120596 induced toxicity in these cells occurs rapidly, in less than 10 minutes. Bovine Serum Albumin Eliminates the Temperature Dependence of PNU 120596 Toxicity and, to a Lesser Degree, P otentiation A ctivity As stated above, an observation made with the initial experiments is that choline and 10 M PNU 120596 treatments were toxic at 37C when the solutions were prepared in DMEM with 10% FBS. This result was curious and led to additional experiments in which choline + 10 M PNU 120596 solutions were prepared in HBSS

PAGE 165

165 supplemented with 10% FBS. As seen in Figure 7 7A and B, the presence of FBS eliminated the temperature dependence of the PNU 120596 toxic ity. Bovine serum albumin is the primary constituent of FBS and serum albumins have previously been shown to potentiate 7 nAChR mediated re sponse s [ 445 ] Therefore, the experiments were repeated in HBSS solutions containing 30 M BSA, the approximate concentration of BSA found in solutions containing 10% FBS [ 446 447 ] Again, the temperature dependence of cytotoxicity induced by PNU 120596 was eliminated (Figure 7 7C and D), suggesting that BSA is the constituent of FBS primarily responsible for the effect. Given that BSA is a non specific carrier of hormones and fatty acids in b lood plasma, it is possible that a substance bound to the BSA is responsible for the effect, rather than BSA itself. Nonetheless, this observation suggests that although, the ability of PNU 120596 to potentiate 7 mediated current at physiological temperature is reduced, some intrinsic factors may exist that confer activity to PNU 120596 under conditions when it would otherwise be inactive. The toxic effect of 100 M choline + 10 M PNU 120596 treatment at 37 ¡ C in HBSS with 30 M BSA was tested for sensitivity to the competitive antagonist MLA, and also the non competitive antagonist mecamylamine to confirm this effect is mediated by 7 nAChR and whether it requires activation of the ion channel (Figure 7 8). As before, 10 nM MLA was unable to completely block the toxic effect of the treatment but 10 minute pre applications and co applications of 100 nM MLA completely reversed the toxicity while applications of MLA 10 minutes or more after the choline and PNU 12 0596 treatment were ineffective. In addition, ten minute pre treatment and co treatment with 100 M mecamylamine was able to partially block the toxicity of choline and PNU 120596 treatment while mecamylamine treatments 10

PAGE 166

166 minutes or more after the treatment had no effect. The IC 50 for mecamylamine was approximately 10 M in experiments performed in oocytes where mecamylamine was co applied with 300 M ACh [ 448 ] In addition, 100 M mecamylamine appears to fully block steady state currents generated by choline and PNU 120596 co ap plication in recent experiments [ 376 ] Although the 100 M concentration of mecamylamine was unable to completely block the toxic effect in these studies, the partial block that was observed combined with the rapid onset of the toxicity in less than 10 minutes is consistent with direct ion channel activity. T he observation that 100 nM MLA completely blocked the toxic effect of choline + PNU 120596 treatment at 37 ¡ C in the presence of 30 M BSA suggests the effect is mediated by the 7 nAChR. The Temperature D ependence of PNU 120596 is Confirmed through Whole Cell Patch Clamp Recordings The whole cell electrophysiology data presented below were collected and analyzed by Dr. Can Peng. Only her data with PNU 120596 that are relevant to the cytotoxicity experiments will be shown here. The basic protocol used in these experiments was to obtain three responses at room temperature (23.5 ¡ C), record three responses at 37 ¡ C, and then reduce the temperature back down to 23.5 ¡ C over a period of 20 minutes. Acetylcholine was co applied with PAM for 3 seconds with 57 sec ond inter stimulus intervals. Prior to presenting the data, it is important to note that above ~30C th e quality of the whole cell recordings almost always deteriorated. Because of this, the parameters used to define an acceptable whole cell recording at 37C were more relaxed than they would be for a typical whole cell recording made at room temperature. Whole cell seals w ith access resistance < 40 megaO h ms, input resistance > 100 megaO hms, and holding current < 700 pA at 37 ¡ C were deemed

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167 acceptable. P rior to the increase in temperature, ac cess resistances were < 15 megaO hms, input resistances were > 1 gigaO hm, and the holding current was between 50 pA and 0 pA. In most cases, if the patch survived the time at 37C, the whole cell parameters improved as temperatures returned near room temperature. In rundown control experiments performed over a period of 20 minutes with no temperature adjustments the amplitude of the responses at the end of the experiment were ~70% of the responses at the beginning ( Figure 7 9A and B). When 1 mM ACh was applied without a PAM, peak currents at 37 ¡ C were 444% of the initial baseline currents recorded at 23.5 ¡ C and they recovered to ~ 75% upon temperature reduction back to 23.5 ¡ C, a full recovery based on the r undown control (Figure 7 9C and D). Thus, it is important to note that in these experiments currents evoked by ACh alone were reduced by approximately 56% at 37 ¡ C. When evaluating the effect of temperature on PNU 120596 potentiation, the ACh evoked respo nses at 37 ¡ C are used as the base line for comparison The deterioration in whole cell recording properties at 37 ¡ C could lead to a reduction in the fidelity of the voltage clamp. It is feasible that the currents evoked in the presence of PNU 120596, give n their size, could contribute to a greater loss of voltage clamp fidelity than may have occu r red with ACh alone. However, the increased temperature had less of an effect on the responses evoked with TQS than on the responses evoked with PNU 120596, relat ive to the initial baseline responses (44 3% vs 11 3% ; TQS data not shown here, but will be included in Williams et al ., in preparation ). This is despite the fact that the absolute magnitude of the responses recorded with TQS was larger than those wit h PNU 120596 (3,080 484 pA vs 1 333 414 pA at 23.5 ¡ C and 1,438 229 vs 104 31 pA at

PAGE 168

168 37 ¡ C). These data suggest that temperature has a unique effect on potentiation by PNU 120596 and that voltage clamp errors are probably not responsible for the di minished currents with PNU 120596 at 37 ¡ C. In addition, the toxicity data provide another form of evidence that PNU 120596 potentiation is reduced at 37 ¡ C. When 10 M PNU 120596 was co applied with 1 mM ACh, potentiated responses at 37 ¡ C were re duced to a greater extent than the response reduction that occurred when ACh was applied alone (Figure 7 10A and B). At 37C PNU 120596 potentiated responses were only 113% (~89% reduction) of the baseline responses obtained initially at 23.5 ¡ C, but reco vered fully when the temperature was returned to 23.5 ¡ C. This result is consis tent with the findings of Sitzia et al 2011 and also correlates well with the temperature dependence of the PNU 120596 cytotoxicity seen above. One hypothesis to explain this phenomenon is that entry into PNU 120596 insensitive desensitized states occurs more readily at 37 ¡ C than at room temperature. Since recently published work suggests that D i states are stabilized by high PNU 120596 concentrations, this hypothesis was tes ted by repeating the temperature experiment with a 10 fold lower concentration of PNU 120596. However, the result with 1 M PNU 120596 was similar to that with 10 M PNU 120596 (Figure 7 10C and D). When 1 M PNU 120596 was co applied with 1 mM ACh, potentiated responses at 37C were 93% (~91% reduction) of the initial responses at 23.5 ¡ C, and then fully recovered when the temperature was returned to room temperature. Since 30 M BSA appeared to eliminate the temperature dependence of PNU 120596 cy to to xicity in a manner that was dependent on 7 nAChR signaling whole cell electrophysiology experiments were performed in the presence of 30 M BSA. W he n

PAGE 169

169 the responses recorded with 30 M BSA at 37 ¡ C were expressed relative to the initial responses obtained at 23.5 ¡ C, the effect of BSA on the normalized peak currents was not statistically signific ant (Figure 7 11A; p>0.05). Ho wever, when the same data were plotted based on the absolute magnitude of the recorded peak currents, the responses recorded in the presence of 30 M BSA at 23.5 ¡ C and 37 ¡ C were significantly larger than responses recorded in the absence of BSA (Figure 7 1 1B; p<0.05 ; see Chapter 3 for statistical methods ). On average, the peak currents evoked by 1 mM ACh and 10 M PNU 120596 co application at 23.5 ¡ C in the absence and presence of 30 M BSA were 1 333 414 pA and 2,951 307 pA, respectively. The average peak currents recorded at 37 ¡ C in the absence and presence of 30 M BSA were 104 31 pA and 422 117 respectively. These data suggest that 30 M BSA potentiates 7 mediated responses, and does so in an additive manner with PNU 120596. Discussion Numer ous lines of in vitro evidence from independent labs suggest that potentiators of 7 mediated signals may produce signals with relevance to living biological systems Although the available in vivo data are relatively limited at the present time, 7 PAMs have been shown to produce measurable effects through behavioral measures of cognitive function and has been shown to reverse auditory gating deficits in drug induced or DBA/2 models when admi nistered to living animals [ 322 ] The se studies suggest that the 7 PAMs possess sufficient pharmacokinetic properties to modulate brain 7 receptors in vivo and suggest that the PAMs are pharmacologically active at physio logical temperature. T he finding that PNU 120596 administration improved auditory gating deficits induced by amphetamin e [ 318 ] and

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170 deficits in the set shifting tasks induced by phencyclidine [ 449 ] may be seen as inconsistent with the temperature dependence of PNU 1 20596 originally reported by Sitzia et al 2011. However, th e viability assays and electrophysiology experiments performed in solutions containing BSA suggest that some intrinsic factor may partially preserve the pharmacological activity of PNU 120596 at 37 ¡ C, and may preserve the activity sufficiently well to cont inue producing biologically relevant signals (in this case, toxic signals). Given that 7 nAChR has an intrinsically low P open and PNU 120596 is such a profoundly powerful potentiator, it seems conceivable that even a modest preservation of PNU 120596 potentiation may be sufficient to produce significant in vitro cytotoxicity at 37 ¡ C and eff ects in vivo More pre clinical in vivo data regarding the activity of PNU 120596 and other 7 PAMs are needed to fully evaluate their potential utility as therapeutics. At any rate, the data presented here suggest that experiments performed at room temp erature should be applied with extreme caution in the interpretation of data generated in vivo At this point it would be entirely speculative to state a mechanism for the apparent temperature dependence of PNU 120596. Therefore, only one brief co mment will be made regarding this matter until further information is available. The putative binding site for PNU 120596, and other PAMs, is in the intrasubunit cavity formed by the four membrane spanni ng helices [ 322 344 ] The increased kinetic energy at 37 ¡ C may alter the protein conformation, fluidity of the membrane and/or the composition of the membr ane at the receptor membrane interface in a manner that disrupts the mechanism of PNU 120596. The functional properties of nAChRs have been shown to be affec ted by interactions with lipids [ 450 ]

PAGE 171

171 One of the purposes of the cytotoxi city experiments was to characterize the in vitro cytotoxicity profile of PNU 120596 over a wide range of agonist and modulator concentrations. In HBSS based experimental solutions and 28 ¡ C incubations, the data suggest that PNU 120596 is toxic in a conce ntration dependent manner and the observation that the magnitude of toxicity decreased between treatments with 10 M PNU 120596 and 30 M PNU 120596 is consistent with the induction of D i states. In contrast, there was no clear dependence of toxicity on choline concentration over a given PNU 120596 concentration. The experiments performed with the 7 antagonists MLA a nd mecamylamine suggest that the onset of PNU 120596 toxicity is rapid. The rapid onset of toxicity could conceivably account for the lack of a clear choline concentration dependence in the toxicity experiments with PNU 120596. Even if D i did eventually accumulate when choline concentrations were high, the cells may have died before a significant accumulation of D i could occur to prevent the toxicity. The finding that co application of 30 M 3mM choline with 10 M PNU 120596 produces toxicity at 37 ¡ C in the presence of FBS is cons istent with the findings of Dinklo et al 2011 and Ng et al ., 2007 and incons istent with the results of Hu et al ., 2009 Both of the published studies that show evidence in favor of in vitro PNU 120596 toxicity were incubated at 37 ¡ C in media containing FBS for 24 hours. Unfortu nately, the methods used by Hu et al 2009 to show the lack of PAM cytotoxicity are vague In addition, the fact that Hu et al ., 2009 f ail ed to directly demonstrate functional 7 receptors in their undifferentiated PC12 cells and cultured cortical neurons is a severe limitation of their study. Furthermore, the fact that there is no dependence on modulator concentration for any of the PAMs tested in this study is curious. How ev er, the

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172 possibility that induction of D i states accounted for the lack of toxicity in these studies still cannot be completely ruled out.

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173 Figure 7 1. Expression of human 7 and ric3 by the HEK h 7/hric3 cell line. A) Expression of human 7 and ric3 mRNA is verified by RT PCR The observed bands for GAPDH, hric3, and h 7 are 516 bp, 346 bp, and 414 bp, as expected based on the primers used. B) Immunoprecip it ation and western blot from cell lysates performed by Clare Stokes and Monica Santisteban Untransfected HEK293 and HEK hric3 cells were negative for h 7 protein. The labeled protein from HEK h 7 and HEK h 7/hric3 cell lysates is an aggregate with molecular weight > 220 kDa. The primary antibodies for 7 were gen erously provided by Dr. Cecilia Gotti (University of Milan, Italy).

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174 Figure 7 2 Labeling of intact HEK h 7/hric3 cells with Alexa Fluor488 bungarotoxin. No labeling was observed for untransfected HEK293, HEK hric3, or HEK h 7 cell lines. In c ontrast, labeling was observed on HEK h 7/hric3 cells in a competitive manner with 1 mM nicotine. Cellular nuclei are stained in blue with DAPI and the Alexa Fluor488 bungarotoxin label is green

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175 Figure 7 3. Saturation binding of [ 125 I] bungaroto xin b inding to intact HEK h 7/hric3 cells A) No specific binding is detected with untransfected HEK293. Values are the mean SEM of 5 6 replicates. B) A saturation binding curve from one representative experiment is shown on the left and the Scatchard transformation of the same data is shown on the right. The average K d and B max values from three independent saturation binding experiments are 991 67 pM and 7.71 x 10 8 1.28 x 10 8 pmol/cell, respectively. S pecific binding is defined as the differe nce between total binding and non specific binding. Non specific binding was determined using 1 M unlabelled bungarotoxin.

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176 Figure 7 4. ACh concentration response relationship and inhibition of currents by MLA from HEK h 7/hric3 cells. A) Concentration response relationship for whole cell peak currents and net charge responses evoked by AC h. The curve for net charge was fit between 1 M ACh and 300 M ACh, denoted by the solid grey boxes. Each point represents the mean SEM from 4 8 cells. Net charge responses were calculated for a period of 1 s following ACh application. B) Inhibition of responses by MLA determined from peak responses. In these experiments increasing MLA concentrations were co applied with 170 M ACh, the EC 50 for peak currents determined in part A. Each point represents the mean SEM determined from 4 6 cells. The holding potential was 70 mV.

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177 Figure 7 5. Temperature and concentration dependence of PNU 120596 cytotoxicity with treatments prepared in HBSS solutions. A) Cytotoxicity profile of 0 30 M PNU 120596 with 0 3 mM choline when treatments were incubat ed at 28 ¡ C /5%CO 2 B) Cytotoxicity profile of 0 30 M PNU 120596 with 0 3 mM choline when treatments were incubated at 37 ¡ C /5% CO 2 The indicates a two tailed p value < 0.05. Each value is the average SEM of 3 5 independent experiments. Two sets of cells were plated from the same passage one day prior to experiments The two sets of cells were treated identically with the same experimental solutions and then one set of cells was immediately placed in a CO 2 incubator set to 28 ¡ C and the other set of cells in an incubator set to 37 ¡ C for 2 hours. U ntransfected HEK293 cells were treated in parallel and were unaffected by all choline and PNU 120596 treatments at 28 ¡ C and 37 ¡ C (data not shown).

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178 Figure 7 6. Sensitivity of the cytotoxic effect of 100 M choline + 10 M PNU 120596 treatment in HBSS at 28 ¡ C to the competitive antagonist MLA. Either 10 nM or 100 nM MLA was added at the time indicated, relative to the toxic 100 M choline + 10 M PNU 120595 treatment. The indicates a two tailed p v alue < 0.05. Values are averages SEM from 3 independent experiments.

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179 Figure 7 7. Elimination of the temperature dependence of PNU 120596 cytotoxicity. A) Cytotoxicity of 0 3 m M choline and 10 M PNU 120596 co applications at 28 ¡ C in HBSS solut ions with 10% FBS. B) Cytotoxicity of 0 3 mM choline and 10 M PNU 120596 co applications at 37 ¡ C in HBSS solutions with 10% FBS. Notably, a factor in FBS ap pears to remove the temperature dependent cytoto xicity seen in Figure 7 5. C) Cytotoxicity of 0 3 mM choline and 10 M PNU 120596 co applications at 28 ¡ C in HBSS solutions with 30 M BSA. D) Cytotoxicity of 0 3 M choline and 10 M PNU 120596 co applications at 37 ¡ C in HBSS solutions with 30 M BSA. The effect of 30 M BSA appears to be very similar to that of FBS. The indicates a two tailed p value < 0.05. Values are averages SEM from 3 independent experiments.

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180 Figure 7 8. Sensitivity of the cytotoxic effect of 100 M choline + 10 M PNU 120596 treatment in HBSS with 30 M BSA at 3 7 ¡ C to the competitive antagonist MLA and the non competitive antagonist memamylamine. 10 nM and 100 nM MLA or 10 M and 100 M mecamylamine were added at the time indicated, relative to the toxic treatment. The indicates a two tailed p value < 0.05. Values are averages SEM from 3 independent experiments.

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181 Figure 7 9. Controls for the whole cell patch clamp recordings illustrating the temperature dependence of PNU 120596 potentiation. These data were collected an d analyzed by Dr. Can Peng. A) Rundown control performed at 23.5 ¡ C. Peak responses to 3 second applications 1 mM ACh were recorded every 60 seconds over a period of 20 minutes. Each point is the average SEM of 15 cells. B) Temperature dependent effects of peak responses evoked by 1 mM ACh. Each point is the average SEM of 6 cells.

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182 Figure 7 10. Temperature dependence of PNU 120596 potentiation of 7 mediated responses. These data were collected and analyzed by Dr. Can Peng. A) Whole cell recordings from HEK h 7/hric3 cells evoked by 3 second co applications of 100 M ACh and 10 M PNU 120596 every 60 seconds over a period of 20 minutes with varied temperatures between 23.5 ¡ C and 37 ¡ C. Each point represents the average SEM of 8 cells. B) Sample data traces recorded at th e indicated temperature. C) Whole cell recordings from HEK h 7/hric3 cells evoked by 3 second co applications of 100 M ACh and 1 M PNU 120596 every 60 seconds over a period of 20 minutes with varied temperatures between 23.5 ¡ C and 37 ¡ C. Each point repr esents the average SEM of 6 cells. D) Sample data traces recorded at the indicated temperature. Responses were measured as peak currents. The responses are normalized to the three initial responses obtained at 23.5 ¡ C

PAGE 183

183 Figure 7 11. Modest preservat ion of PNU 120596 potentiation at 37 ¡ C in solutions containing 30 M BSA. These data were collected and analyzed by Dr. Can Peng. A) Whole cell recordings from HEK h 7/hric3 cells evoked by 3 second co applications of 100 M ACh and 10 M PNU 120596 in the absence or presence of 30 M BSA with varied temperatures betwe en 23.5 ¡ C and 37 ¡ C. The data plotted in the absence of BSA are the same data shown in Figure 7 10A Responses were normalized to the initial responses obtained at 23.5 ¡ C Each point in the presence of 30 M BSA represents the average SEM of 9 cells. Responses were measured as peak currents. B) The same data shown in panel A expressed as peak current magnitude rather than normalized responses. C ) Sample data traces recorded at the indicated temperature.

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184 Table 7 1. 10 90% rise times and rise slope s with increasing concentrations of ACh [ACh], M 10 90% Rise time (ms) 10 90% Rise slope (pA/ms) 30 74.6 30.8 0.195 0.081 100 22.3 7.20 1.34 0.465 300 9.86 1.45 22.7 6.90 1000 2.61 0.26 47.2 12.4 3000 1.84 0.53 294 105

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185 CHAPTER 8 SUMMARY AND CONCLUSI ONS The work presented in this dissertation has provided insights regarding the activation, desensitization, and potentiation of 7 nAChRs that will hopefully b e useful in optimizing the therapeutic targeting of this receptor. Though 7W149 and 7W55 are both highly conserved aromatic residues found within the agonist binding sites, they appear to play different roles in receptor activation by agonist. The W55 residue is important for regulating the subtype selectivity of benzylidene anabaseine compounds whereas the W149 residue is less able to tolerate mutation. Although the 7 nAChR has five agonist binding sites, effective activation occurs with low fractio nal occupancy. In contrast, occupancy of all five binding sites appears to actually promote inactivation of the receptor, rather than activation. Characterization of the 7 PAM PNU 120596 has revealed that at least two forms of 7 desensitization exist: the well known rapid and concentration dependent desensitization that is characteristic of 7, and desensitized states that are stable in the presence of the modulator PNU 120596. The in vivo implications of D i states have not been investigated. On one h and, these states may prove to be a real limitation to the usefulness of PAMs like PNU 120596. On the other hand, D i states may provide an intrinsic safety mechanism against over stimulation. The reported temperature dependence of PNU 120596 potentiation has been confirmed through electrophysiology and in vitro cytotoxicity experiments with a novel cell line stably expressing human 7 and ric3. However, the presence of certain factors, such as BSA, may partially preserve the activity of PNU 120596 at physiological temperature to an extent that the temperature dependence of PNU 120596 induced in vitro cytotoxicity is eliminated.

PAGE 186

186 A general qualitative model for the 7 nAChR is suggested based on the data presented here and years of work performed in Dr. Roger Papke's laboratory. The early single channel studies of muscle type nAChR first identified two distinct open states; isolate d short lived openings and longer lived openings that occurred in groups or "bursts". As agonist concentrations were increased, the probability of long lived openings also increased. However, short lived openings continued to occur even under conditions that would saturate the agonist binding site s [ 412 ] Under normal conditions, 7 nAChRs may onl y open to the short lived open state (O*) that is analogous to the short lived type of open state observed predominately with singly liganded heteromeric nAChR; the longer lived open state (O') is replaced by the D s state. The effect of high agonist occup ancy is to stabilize the D s state, similar to the way high agonist occupancy stabilizes the long lived open state in heteromeric nAChRs. This explains the observation that high fractional occupancy of the 7 agonist binding sites appear s to be functionally negative (see Figure 7 4; [ 229 ] ) The modulation o f the 7 D s state by PNU 120596 appears to be through stabilization of intrinsic states of the channel, rather than creation of new conducting states, since PNU 120596 has no effect on ion selectivity and little, if any, effect on channel conductance [ 318 ] Basic models of nAChRs are required to account for closed states, open states, and desensitized states as well as the effects of agonist binding and the relative occupancy of these states. The model in Figure 8 1 describes the functional states of the receptor based on hypothetical relative free energy levels and intervening energy barriers through a matrix of increasing occupancies at both agonist and PAM binding sites. Vertical distances represent absolute differences in free energy, or stability, of the

PAGE 187

1 87 various states under equilibrium conditions. The relative energy levels and heights of the energy barriers are proportionate to the logs of the transition rate constants in standard Markov models. It is important to emphasize that the model is intended to only represent qualitative characteristics of the data obtained with the agonist ACh. To simplify comparisons between the schematics for the various levels o f agonist occupancy, the resting closed states were set to the same level. However, it should be appreciated that the average free energy of the resting bound state for each schematic probably increases (becomes less stable) in a step wise manner as agoni st occupancy increases. The shallow energy well assigned to the short lived open state characteristic of 7 (O*) is consistent with the brief opening observed in single channel recordings, and the high energy barriers into the O* state are consistent with the low P open of 7. Transition from C to O* will never occur with high probability, but is most likely p rior to saturation of the agonist binding sites. The stability (represented by vertical displacements) of the D s and D i states relative to C increases with agonist occupancy, and the equilibrium between D s and D i will most strongly favor D i at the highest levels of agonist and PAM occupancy. Single channel openings evoked by ACh in the presence of PNU 120596 can occur as extremely long bursts or groups of openings separated by very short closures. The D s state represents the rapid concentration dependent form of desensitization unique to 7, and the effect of PNU 120596 may be to convert or connect the D s state into the longer lived open state(s) ( O' ) The short intraburst closures, many of which appear as subconductances, may represent the reverberation between D s and O' in the presence of PNU 120596. The O' state(s) may be analogous

PAGE 188

188 to the long lived open states of doubly liganded heteromeric receptors so that conversions between D s to O' are the primary mechanism through which PNU 120596 reconciles th e peak current and net charge ACh concentration response curves of 7 receptors (see Figure 6 1) Entry into D i states primarily occurs through activated channel states, and the barrier heights between O'/D s and D i states are reduced at high occupancy levels. Therefore, strong ion channel activation promotes D i and a p rimary effect of high PAM occupancy is to stabilize D i The stabilization of D i by high levels of both agonist and PAM occupancy results in a window where maximal potentiation occurs within the low intermediate range of both agonist and PAM occupancy. The 7 nAChR has been hotly pursued as a therapeutic target in the last 20 or so years. Tremendous progress has been made in identifying ligands that selectively activa te and potentiate the receptor, yet the optimal approach to pharmacologically target the re ceptor for specific therapeutic purposes is still not understood. Using structurally related agonists that activate the receptor with varying efficacy, it was shown that channel activation is important for improving performance in inhibitory avoidance beh avioral tasks in rodents [ 291 ] However, the most efficacious 7 agonists are not always the most e ffective at producing protective or cognitive enhancing effects in a number of studies. For example, GTS 21 is a partial agonist of 7, yet it has been documented to protect PC12 cells from trophic factor deprivation [ 227 275 451 ] cultured neurons from glutamate induced cytotoxicity [ 276 ] and 7 expressing cell lines from amyloid b eta fragments [ 452 453 ] In addition, GTS 21 has been shown to re duce neuronal cell loss in vivo after ischemic injury or lesions to the nucleus basalis [ 451 ] and has been shown to preserve functional responses of 7 function in hippocampal

PAGE 189

189 interneurons following disruption of cholinergic input by lesioning the fimbria fornix [ 454 ] Furthermore, GTS 21 has been shown to improve attention and memory related tasks in humans [ 455 ] Int e re stingly, GTS 21 is effective at stabilizing the 7 receptor in non conducting desensitized states [ 327 376 ] Together these observations lead to the hypothesis that there may be more to 7 than activation of the ion channel. Perhaps 7 is capable of transmitting intracellular metabotropic signals from non conducting but non e theless functional states. A proteomics study has indicated that many proteins co assemble in complexes with nAChR, including numerous mediators of intracellular signal transduct ion [ 456 ] This idea is still very much in its infancy and others have in fact dec lared desensitized states of ligand gated ion channels to be "more of an experimental nuisance than a physiolo gically interesting phenomenon" [ 65 ] but th ere have been some observations that 7 mediated signal transduction occurs in non neuronal cells even though ion channel activity is not detectable [ 457 458 ] We have shown that different agonists preferentially stabilize specific conformations (i.e. D s or D i ) using PNU 120596 as a probe for functional states [ 376 ] Furthermore, the same ligand may be able to preferentially stabilize specific functional states depending on its binding orientation [ 380 ] Given that the stability of the non conducting conformations appears to be much greater than the stability of the activ ated ion conducting states t he design and discovery of ligands that specifically stabilize desired receptor c onformations could be important to 7 drug development efforts in the future. However, figuring out exactly what the desired conformational states are in order to produce a desired effect is a m onumental task that lies ahead.

PAGE 190

190 Despite the identification and initial characterizing of 7 PAM s including PNU 120596, the current knowledge of the limiting factors for PAM based therapy and the most desirable functional characteristics of PAMs as therapeutic agents is incomplete. Indeed there are more questions than answers. For example, are ther e different conditions when either a type I or type II PAM might be most advantageous? To what degree might a type II PAM disturb native temporal characteristics of a neuronal circuit? Can a PAM alter the native channel kinetics and still provide an acce ptable therapeutic index? Are the most efficacious PAMs most desirable? Can 7 PAMs induce cytotoxicity due to high levels of channel mediated calcium flux? What are the limiting factors of allosteric potentiation, and under what condition is the potenti ation optimized? Does the ability of a PAM to modulate responses change over significant amounts of time? Are the known 7 PAMs sufficiently selective to avoid undesired effects? Would sufficient endogenous acetylcholine be present in patients with dege nerated cholinergic neurons for effective positive allosteric modulation? Will PAMs retain sufficient pharmacological activity at physiological temperature to be useful? Some of these questions have begun to be answered, but clearly, the search for under standing and identifying characteristics of an ideal 7 PAM continues.

PAGE 191

191 Figure 8 1 Proposed qualitative models for the activation, desensitization, and modulation of 7 nAChR. This figure was prepared together with Dr. Roger Papke. In the absence of any PNU 120596 binding, the primary effect of agon ist binding is to shift the equilibrium between the conformational states from the resting closed state toward the desensitized states, D s, which is sensitive to destabilization by PNU 120596, and D i which is insensitive to the activating effects of PNU 1 20596. The lower two rows include a new long lived open state O', promoted by the binding of PNU 120596 and possibly connected to a modified form of the D s state. Both agonist and PAM binding dynamically regulate the balance between D s and D i M aximal ion channel activity occurs with intermediate levels of both agonist and PAM occupancy.

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192 REFERENCES [1] Schuetze SM. The discovery of the action potential. Trends Neurosci 1983;6:164 8. [2] Lopez Munoz F, Alamo C. Historical evolution of the neurot ransmission concept. J Neural Transm 2009;116:515 33. [3] Bennett MR. The early history of the synapse: from Plato to Sherrington. Brain Res Bull 1999;50:95 118. [4] Cobb M. Timeline: exorcizing the animal spirits: Jan Swammerdam on nerve function. Nat R ev Neurosci 2002;3:395 400. [5] Verkhratsky A, Krishtal OA, Petersen OH. From Galvani to patch clamp: the development of electrophysiology. Pflugers Arch 2006;453:233 47. [6] Cajavilca C, Varon J, Sternbach GL. Resuscitation great. Luigi Galvani and the foundations of electrophysiology. Resuscitation 2009;80:159 62. [7] Piccolino M. Animal electricity and the birth of electrophysiology: the legacy of Luigi Galvani. Brain Res Bull 1998;46:381 407. [8] Goldensohn ES. Animal electricity from Bologna to Bos ton. Electroencephalogr Clin Neurophysiol 1998;106:94 100. [9] Kettenmann H. Alexander von Humboldt and the concept of animal electricity. Trends Neurosci 1997;20:239 42. [10] Hoff HE, Geddes LA. The rheotome and its prehistory: a study in the historical interrelation of electrophysiology and electromechanics. Bull Hist Med 1957;31:327 47. [11] Seyfarth EA. Julius Bernstein (1839 1917): pioneer neurobiologist and biophysicist. Biol Cybern 2006;94:2 8. [12] Piccolino M. Fifty years of the Hodgkin Huxley era. Trends Neurosci 2002;25:552 3. [13] Edidin M. Lipids on the frontier: a century of cell membrane bilayers. Nat Rev Mol Cell Biol 2003;4:414 8. [14] Nilius B. Pflugers Archiv and the advent of modern electrophysiology. From the first action potential to patch clamp. Pflugers Arch 2003;447:267 71.

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230 BIOGRAPHICAL SKETCH Dustin Kyle Williams was born November 12, 1982 in Logan, Utah to Kevin and Kathy Willia ms. Dustin grew up in Boise, Idaho and graduated from Centennial High School in 2001. After high school, Dustin completed a two year mission for the Church of Jesus Christ of Latter day Saints in Rio Grande do Sul, Brazil from January 2002 to December 20 03. Upon returning from Brazil, Dustin completed one year of college at Boise State University before transferring to Brigham Young University Hawaii where he completed an undergraduate degree in Biochemistry in June 2007. Dustin met his wife, Julie, whi le they were both students in Laie, Hawaii. Dustin started graduate school at the University of Florida in August 2007 with an elementary interest in understanding how neuronal activity produces behavior. He joined the laboratory of Dr. Roger Papke, wher e he learned electrophysiological methods to study nAChRs an important family of ion channels expressed in the central and peripheral nervous systems.