[3H]-Dihydroerysovine As a Molecular Probe of the Resting State Alpha4beta2 Nicotinic Acetylcholine Receptor

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[3H]-Dihydroerysovine As a Molecular Probe of the Resting State Alpha4beta2 Nicotinic Acetylcholine Receptor
Chen, Chao
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[Gainesville, Fla.]
University of Florida
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Master's ( M.S.)
Degree Grantor:
University of Florida
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Medical Sciences
Committee Chair:
Kem, William R.
Committee Members:
Silverman, David N.
Bruijnzeel, Adriaan Willem
Baker, Stephen P.
Purich, Daniel L.
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Agonists ( jstor )
Alkaloids ( jstor )
Binding sites ( jstor )
Cell membranes ( jstor )
Ligands ( jstor )
Nicotinic receptors ( jstor )
Oocytes ( jstor )
pH ( jstor )
Rats ( jstor )
Receptors ( jstor )
Medicine -- Dissertations, Academic -- UF
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Medical Sciences thesis, M.S.


[3H]-DIHYDROERYSOVINE AS A MOLECULAR PROBE OF THE RESTING STATE ALPHA4BETA2 NICOTINIC ACETYLCHOLINE RECEPTOR The ?4?2 neuronal nicotinic acetylcholine receptor (nAChR) is the most abundant heteromeric nAChR in the mammalian brain and mediates much of the dopamine-releasing and cognition-enhancing effects of nicotine. Currently, many agonists and partial agonists at this nAChR are used as molecular probes or as drug candidates for treatment of certain neurodegenerative or neurodevelopmental diseases in which nAChRs are dysfunctional. In contrast, the ?4?2 antagonists have been less well studied and there is a need for more improving their nAChR selectivity. Coral bean (Erythrina) alkaloids have long been known for their curare-like effects on peripheral nAChRs. We have prepared a potent antagonist, dihydroerysovine (DHSOV), from the Erythrina alkaloid erysovine, and obtained a highly radioactive (tritiated) form of this compound to use as a molecular probe. DHSOV has a higher affinity at ?4?2 nAChR than the other known ?4?2 antagonists such as dihydro-?-erythroidine (DH?E) and erysodine. Using a saturation binding assay, I determined the affinities of DHSOV for nAChRs present in rat brain membrane (RBM) and in membranes from TSA-201 cells specifically expressing ?4?2 or ?4?5?2 receptors. The data suggested that the normally expressed ?4?2 receptor and another, as yet to be identified receptor have high affinity to DHSOV. My data also showed that DHSOV binding to the ?4?2 receptor is pH dependent at pH 7.4-9.0; affinity of binding decreased with an increase in pH. The ionized form of DHSOV seems to bind with high affinity at the ?4?2 receptor relative to the unionized form. In addition, the displacement of DHSOV binding in RBM by several common nAChR ligands indicates that [3H]-DHSOV is a useful molecular probe for measuring the affinity of an agonist for the ?4?2 receptor resting state. ( en )
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Thesis (M.S.)--University of Florida, 2009.
Adviser: Kem, William R.
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by Chao Chen.

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2009 Chao Chen 2


ACKNOWLEDGMENTS To begin with, I sincerely acknowledge the in valuable guidance provided by my advisor, Dr. William Kem. He is a kind person, a patient t eacher, and an excellent scientist. I am glad I had the opportunity to be his student. I would also like to thank my supervisory committee, Dr. Stephen Baker, Dr. Adriaan Bru ijnzeel, Dr. Daniel Purich, and Dr. David Silverman, for their guidance, support and evaluation of this thes is. Next, I would also like to thank the Pharmacology Department staff for their gracious help during my entire graduate study. I would like to express my gratitude to the past and pr esent members of the Kem lab, especially to Dr. Ferenc Soti for the isolation and syntheses of the crucial compounds, Dr. Jian Zuo for her instruction in mutagenesis experiments, a nd Dr. Hong Xing for her support in oocyte experiments. I extend many thanks to Dr. J on Lindstrom for gracious ly providing the TSA201 cells used in binding assays. I also thank the people in the Katritzky lab for their help in molecular modeling. Finally, I would like to expr ess my eternal gratitude to my parents and loving family. They have always encouraged me to continue the difficult journey. 3


TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF TABLES................................................................................................................. ..........5 LIST OF FIGURES.........................................................................................................................6 ABSTRACT.....................................................................................................................................7 CHAPTER 1 INTRODUCTION................................................................................................................. ...9 Nicotinic Acetylcholine Receptors...........................................................................................9 Acetylcholine-Binding Protein...............................................................................................10 Erythrina Alkaloids................................................................................................................11 Purpose of Thesis Research....................................................................................................1 2 2 MATERIALS AND METHODS...........................................................................................14 Materials.................................................................................................................................14 Rat Brain Membranes............................................................................................................ .14 TSA201 Cell Membranes.......................................................................................................15 Radioligand Binding Assays...................................................................................................15 Binding Assay Data Analysis.................................................................................................16 Site-directed Mutagenesis of Mutant 2 Subunit...................................................................17 In vitro Synthesis of mRNA and Oocyte Expression.............................................................18 Electrophysiology and Data Analysis.....................................................................................18 3 RESULTS...................................................................................................................... .........20 [3H]-DHSOV Saturation Binding in Rat Brain Membrane (RBM)........................................20 Influence of pH on [3H]-DHSOV Binding in RBM...............................................................20 Displacement of [3H]-DHSOV Binding in RBM by Agonists and Antagonists....................21 [3H]-DHSOV Saturation Binding in TSA-201 Cells Expressing 4 2 nAChRs...................22 Threonine 84 of 2 is Involved in DH E Binding.................................................................22 4 DISCUSSION................................................................................................................... ......33 LIST OF REFERENCES...............................................................................................................38 BIOGRAPHICAL SKETCH.........................................................................................................42 4


LIST OF TABLES Table page 3-1 The specific binding of [3H]-DHSOV at pHs 7.4, 8.2 and 9.0 to rat brain membranes..........25 3-2 Ki values of some common nAChR ligands as determined by displacement of [3H]DHSOV and other radioligands selective for the 4 2 nAChR........................................26 3-3 Comparision of Hill slopes and Kis obtained from displacement experiments when two different concentrations of [3H]-DHSOV were tested.......................................................27 5


LIST OF FIGURES Figure page 1-1 Structures of several Erythrina alkaloid analogs.....................................................................13 3-1 [3H]-DHSOV binding in rat brain membranes........................................................................24 3-2 [3H]-DHSOV saturation binding in TSA-201 cel ls at concentrations of 0.05-20 nM............28 3-3 [3H]-DHSOV saturation binding in TSA-201 cells at concentrations of 0.05-20 nM after 20 M nicotine treatment of TSA-201 cells over night.....................................................29 3-4 [3H]-DHSOV saturation binding in TSA cells expressing 4 5 2 nAChRs at the concentration of 0.05-20 nM..............................................................................................30 3-5 ACh dose-response curves for wild type, 2T84A, 2T84S and 2T84Y 4 2 receptors.....31 3-6 DH E inhibition curves for wild type, 2T84A, 2T84S and 2T84Y 4 2 receptors.........32 6


Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science [3H]-DIHYDROERYSOVINE AS A MOLECULAR PROBE OF THE RESTING STATE AL PHA4BETA2 NICOTINIC ACETYLCHOLINE RECEPTOR By Chao Chen August 2009 Chair: William R. Kem Major: Medical Sciences The 4 2 neuronal nicotinic acetylcholine recep tor (nAChR) is the most abundant heteromeric nAChR in the mammalian brain and mediates much of the dopamine-releasing and cognition-enhancing effects of nicotine. Curren tly, many agonists and par tial agonists at this nAChR are used as molecular probes or as drug candidates for treatment of certain neurodegenerative or neurodevelopmental diseases in which nAChRs are dysfunctional. In contrast, the 4 2 antagonists have been less well studied and there is a need for more improving their nAChR selectivity. Coral bean ( Erythrina ) alkaloids have long been known for their curarelike effects on peripheral nAChRs. We have prep ared a potent antagonist, dihydroerysovine (DHSOV), from the Erythrina alkaloid erysovine, and obtaine d a highly radioactive (tritiated) form of this compound to use as a molecular probe. DHSOV has a higher affinity at 4 2 nAChR than the other known 4 2 antagonists such as dihydro-erythroidine (DH E) and erysodine. Using a saturation binding assay, I de termined the affinities of DHSOV for nAChRs present in rat brain membrane (RBM) and in membranes from TSA-201 cells specifically expressing 4 2 or 4 5 2 receptors. The data suggested that the normally expressed 4 2 receptor and another, as yet to be identified receptor have high affinity to DHSOV. My data also showed that DHSOV binding to the 4 2 receptor is pH dependent at pH 7.4-9.0; affinity of 7


binding decreased with an increase in pH. The ionized form of DHSOV seems to bind with high affinity at the 4 2 receptor relative to the unionized form. In addition, the displacement of DHSOV binding in RBM by several common nAChR ligands indicates that [3H]-DHSOV is a useful molecular probe for measuring the affinity of an agonist for the 4 2 receptor resting state. 8


CHAPTER 1 INTRODUCTION Nicotinic Acetylcholine Receptors Nicotinic acetylcholine receptors (AChRs) be long to the prototypic Cys-loop family of ligand-gated ion channels (LGI Cs), which also includes 5-HT3, glycine and GABAA/C receptors. These receptors all possess a 13 amino acids signature sequence within a Cys-loop (Connolly and Wafford, 2004). The endogenous neurotransmitter acetylcholine (ACh) binds to one or more binding sites on the receptor and causes a confor mational change that leads to channel opening and ion flux. All nAChRs are composed of five homologous subunits organized like barrel staves around a central ion channel. Currently, 17 AChR subunit genes have been cloned and identified, including five muscle subunits ( 1, 1, 1, and ) and 12 neuronal subunits ( 210 and 24). Each subunit has four transmembrane (TM) segm ents, a long extracellula r N-terminal domain, an intracellular loop between TM3 and TM4 and a short C-terminal domain. TM2 is involved in shaping the lumen of the pore, and five TM2s form the ion channel pore (Karlin, 2002). Different combinations of these subunits result in a wide variety of s ubtypes of nAChR subtypes which exhibit different physiological and pharm acological properties (Albuquerque et al., 2009). The heteromeric 4 2 and homomeric 7 nAChRs are the two most abundant receptor subtypes in the mammalian central nervous system (CNS). The 7 nAChR has a very high affinity for bungarotoxin, while 4 2 receptors have a very high affinity for cytisine and nicotine (Gotti et al., 2006). It has been found that nAChRs are associat ed with several CNS diseases such as Alzheimers disease (AD), Parkinsons disease, autosomal dominant noc turnal frontal lobe epilepsy (ADNFLE), schizophrenia, attention defi cit hyperactivity disorder (ADHD) and tobacco 9


addiction (Arneric et al., 2007; Bertrand et al., 2002; Lindstrom, 1997; Wilens and Decker, 2007). The association of particular receptor subtypes with different diseases has stimulated the pharmaceutical development of subtype-selectiv e ligands. Some nAChR ligands have been shown to have therapeutic effects for these associated diseases. For example, it has been found that treatment with nAChR agonist s can alleviate some of the clinical symptoms in the early stages of Alzheimers disease (N ewhouse et al., 2001). GTS-21, an 7 partial agonist, has been shown to enhance cognition in humans (Freedman et al., 2008). Rowland et al. (2008) suggested that nicotine analogs with 4 2 nAChR partial agonist and an tagonist efficacies can inhibit nicotine self-administration in rats (Rowland et al., 2008). Varenicline, an 4 2 partial agonist (Coe et al., 2005), was approved in 2006 by FDA fo r use in the treatment of smoking addiction. Acetylcholine-Binding Protein Acetylcholine-binding protein (AChBP) has become a very useful molecular model for analysis of the ligand interaction with nAChRs since its crystal stru cture was reported in 2001 (Brejc et al., 2001). AChBP is a soluble protein expressed by g lia cells in several mollusks including the fresh water snail Lymnaea stagnalis In mollusks it modulates synaptic transmission in an acetylcholine-dependent manne r (Smit et al., 2001). This protein is a stable homopentamer composed of five 210-residue su bunits which align with the N-terminal domain of various LGICs, but mo st closely with nAChR subunits. Brejc et al. (2001) confirmed that the ligand-binding site is composed of loops A, B and C of the -subunit (the principal part) and loops D, E and F of the -strands of the adjacent subunit (the complementary part). These six loops form a cavity that accommodates the binding ligand. Loop C, containing Tyr185, the double cysteine 187-188 and Tyr192, is an important part of the binding site. When full agonists bind to AChBP, loop C closes over the ligand and this somehow contributes to receptor activation (Taylor et al., 2007). 10


Erythrina Alkaloids Erythrina is a genus of bushes (subtr opical) and trees (tropical) distributed worldwide. The alkaloids extracted from this plant have long been known for their curare-like effects on peripheral nAChRs (Lehman, 1936). Recently, it was found that the water-alcohol extract from Erythrina flower petals also produces anxiolytic -like effects (Onusic et al., 2003). The Erythrina alkaloids share a common heterocyclic ring scaffold. DH E is a well-known competitive antagonist that shows preferential affinity for the 2 subunit containing nAChRs. DH E is semisynthesized from natural -erythroidine by conver ting the two conjugated double bonds in the A and B rings of -erythroidine to one double bond situated between them. This conversion causes an eight-fold affinity increase in binding affinity at the rat 4 2 receptor relative to erythroidine (Wildeboer, 2005). Erysodine is another Erythrina alkaloid which has been identified as a high affinity competitive antagonist for 4 2 nAChR. It was reported that erysodine had an 800-fold greater affinity for rat 4 2 than 7 nAChR (Decker et al., 1995). Interestingly, erysovine, the isomer of erysodine, displays a higher affinity for 4 2 receptor compared with erysodine, 23-fold more for rat 4 2 receptor and 8.9-fold more for human 4 2 receptor (Wildeboer, 2005). So, these compounds can be arranged according to their affinities for 4 2 receptor from low to high: erythroidine < DH E < erysodine < erysovine (Figure 1). Dr. Wildeboer studied the structure-activity re lationship of these plant alkaloids for 4 2 nAChR through characterization of the receptor bi nding and functional pr operties of various Erythrina alkaloid analogs. Comparing the affinities of 3-desmethoxy-erythroidine with -erythroidine and comparing the affinities of erysovine (c ontaining a 3-methoxy group) with erysoline (containing 3-hydroxy group), she concluded that a methoxy group at 3-position is important for high affinity binding at the 4 2 nAChR. By comparing N-methyl-erythroidine with erythroidine, it was also shown that methylation of the nitrog en group greatly reduced activity 11


binding at the 4 2 nAChR. The D-ring 15-OH of DHSOV was also shown to affect 4 2 nAChR binding (Wildeboer, 2005). Beers and Reich (1970) proposed that the di stance between the potential H-bond acceptor group and the cationic nitrogen atom present in many nAChR ligands including agonists and antagonist is 5.9 They also noticed that the oxygen atoms on ring A (3-MeO group) and D (lactone group) in -erythroidine are both 5.9 from the nitrogen atom (Beers and Reich, 1970). Hider et al. (1986) determined the crystal structure of -erythroidine and speculated that structural features that determ ine its antagonist property were lo cated within a 3 radius around the nitrogen atom (Hider et al ., 1986). Crystallography of the AC hBP occupied with different nicotinic ligands and mutagenesi s studies also support the view that the ligand cationic N-H contacts makes a H-bond with carbonyl group at Trp149 in the subunit (Celie et al., 2004). Harvey et al. (1996) identified some of the determinants of DH E sensitivity on the rat nAChR subunit by preparing and testing a series of chimeric 3 2 and 3 4 nAChRs containing mutated subunits. They reported that th reonine at position 59 in the 2 subunit is a critical residue for the interaction of DH E with 3 2 and 3 4 receptors. Purpose of Thesis Research Because there are few antagonists selective for 2-subunit containing nAChRs, the present study has focused on a new Erythrina alkaloid dihydroerysovine (DHSOV) synthesized from erysovine and explored the possibility that [3H]-DHSOV might be a usef ul molecular probe for investigating 2-subunit containing receptors. The receptor binding of [3H]-DHSOV was measured at different pHs to explore the influence of [3H]-DHSOV ionization on ligand affinity for rat nAChRs. The affinities of a variety of 4 2 receptor ligands was measured by displacement of either antagonist ([3H]-DHSOV) or agonist ([3H]-cytisine) radioligands. Because rat brain membranes showed a subpopulation of very high affinity binding sites for [3H]-DHSOV, 12


I tried to identify the nature of this brain receptor subtype. Finally, I examined the importance of threonine on loop D of human 2 subunit for Erythrina alkaloid binding to 4 2 receptor. Figure 1-1. Structures of several Erythrina alkaloid analogs. (A) -erythroidine; (B) dihydroerythroidine (DH E); (C) erysodine; (D) erysovine; (E) dihydroerysovine. They have a common heterocyclic ring system. DH E shown in B is semi-synthesized from erythroidine shown in A. Erysodine shown in C and erysovine shown in D are isomers. Dihydroerysovine shown in E is semi-synthesized from erysovine. 13


CHAPTER 2 MATERIALS AND METHODS Materials Dr. Ferenc Soti in the Kem laboratory s ynthesized dihydroerysovine from natural erysovine that had been pur ified by Dr. Kem using HPLC. The compounds structure was confirmed by NMR and mass spectrometry. This synthetic procedure and HPLC purified erysovine was utilized by a commercial vendor ViTrax Radiochemicals (Placentia, CA), to prepare [3H]-dihydroerysovine (21.4 Ci/mmol). [3H]-cytisine (35.6 Ci/mmol) was purchased from PerkinElmer Life and Analytical Scien ce (Boston, MA). Unless otherwise mentioned, all other chemicals and solvents were purchased fr om Fisher Scientific (Atlanta, GA), Tocris Bioscience (Ellisville, MO), or Sigma-Aldrich (St. Louis, MO). Human embryonic kidney cells (TSA201 cell line) expressing either the human 4 2 or 4 5 2 nAChR were graciously provided by Jon Lindstrom (University of Pennsylvania, Philadelphia, PA). Rat Brain Membranes Frozen Sprague-Dawley male rat brains we re purchased from Pel-Freez Biologicals (Rogers, AR). The rat brain membrane was prepared as described previously (Marks and Collins, 1982) with some modifications (Kem et al., 2004). Frozen rat brai ns were homogenized in Tris binding saline (50 mM Tris buffer, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1mM MgCl2, pH 7.4), then centrifuged at 10,000 rpm for ten minutes. The resulting pellet was homogenized again in fresh Tris binding saline (TBS). The amount of membrane protein was measured using the bicinchoninic acid (BCA) protein assay reagent kit from Pier ce (Rockford, IL). The homogenized and washed brain me mbranes were stored at -85 oC before use. In binding experiments, the volume of rat brain membra nes needed was calculated from the protein concentration. 11 mg rat brain me mbranes were centrifuged and su spended in 11 ml Tris binding 14


saline containing 2 mg/ml of bovine serum albumin (BSA) to reduce non-specific binding. 200 l of homogenized rat brain prot ein was used per tube for radi oligand binding experiment in a final volume of 500 l. TSA201 Cell Membranes TSA201 cells were maintained according to Kuryatov et al. (Kuryatov et al., 2005). Each 500 ml of culture medium was composed of 437.5 ml of Dulbeccos Modified Eagle Medium (DMEM), 5 ml of fetal bovine serum, 5 ml of L-Glutamine (200mM), 5 ml of penicillinStreptomycin (10,000 units/ml), 2.5 ml of Zeocin (100 mg/ml) and 0.3 mg Geneticin. All of these ingredients were purchase d from Invitrogen (Carlsbad, CA ). The cells were kept in a humidified incubator with 5% CO2 at 37 oC. After reaching >80% confluence, cells were removed from the culture flasks w ith a disposable cell scraper and pl aced in 6 ml of Tris binding saline (pH 7.4). The cells pellet was obtained by centrifugation, and then suspended in 12 ml of Tris binding saline. They were then homogenized in the same ma nner as the rat brain membranes. The amount of membrane protein wa s also assessed using BCA prot ein assay kit. In radioligand binding experiments, 100 g of homogenized ce ll membrane protein was added per tube containing in a final volume of 500 l. Radioligand Binding Assays The [3H]-dihydroerysovine binding assays were performed unde r conditions identical with those used to measure [3H]-cytisine binding (Flores et al., 1992). All tested compounds in displacement binding experiments were first dissolved in 10 or 100 mM concentrations in the Tris binding saline. These stock solutions were fu rther diluted in Tris bi nding saline containing 2 mg/ml of bovine serum albumin to produce the desired concentrations. The binding mixture was incubated in a final volume of 500 l for 4 hours at 0-4 oC to reach binding equilibrium. A final concentration of 1 mM nicotine was used to measure nonspecific binding of each radioligand. 15


After incubation, membranes with bound radioligand were rapidly filtered with a Brandel cell harvester (Gaithersburg, MD) on Whatman GF/C gla ss fiber filters that had been presoaked in 0.5% polyethylenimine for 45 min to reduce non-s pecific binding. The samples were washed three times with 3-5 ml of ice-cold Tris bind ing saline. Filters contai ning radioligand were transferred in 7 ml scintillation tubes and placed in 3 ml of 30% ScintiSafe scintillation fluid. Radioactivity was counted on a Beckman LS6500 liquid scintillation co unter (Fullerton, CA) for 5 minutes per sample after samples were so aked over night in scin tillation fluid. Each concentration in the radio ligand binding assay was pe rformed in quadruplicate. The pH dependence experiments were performe d using the Tris binding saline at pHs 7.4, 8.2 or 9.0. BSA and nicotine were dissolved in th e desired pH Tris bind ing saline. The other procedures were the same as the normal binding e xperiments described in the above. The pH was measured in several tubes to assure that it was as desired. Binding Assay Data Analysis Data from radioligand binding assays were an alyzed using GraphPad Prism software (San Diego, CA). Specific binding was termed as the difference between the total binding and nonspecific binding. Saturation assa ys were analyzed by nonlinear regression with a one site binding ( XK XB Yd max) or two sites binding ( XK XB XK XB Yd d 2 2max 1 1max). X axis represents the concentration of radioligand and Y axis represents the specific binding. The [3H]dihydroerysovine Kd and Bmax values for the membrane preparation were thereby obtained. The one site binding analysis was used unless the two site binding s howed a better fit ( p < 0.05 by F test). In order to better visualize the change of Kd or Bmax, a Scatchard plot could be derived by data transformation in GraphPad Prism soft ware. The displacement binding assays were analyzed using a sigmoidal dose response with variable slope 16


( Hillslope XICBottom Top BottomY ) (log50101 where X is the logarithm of concentration of unlabeled compounds; Y is the normalized specific binding; T op = Y value at the top plateau of the curve; Bottom = Y value at the bottom plateau of the curve). Based on IC50 calculated from the above equation, all Ki values were obtained using the Cheng-Prusoff equation ( d iK ligand IC K ][ 150 where [ligand] = free radioligand concentration). Site-directed Mutagenesis of Mutant 2 Subunit The plasmid vector pSP64 (PolyA) containing the human nAChR 2 subunit cDNA was graciously provided by Professor Jon Lindstrom (Uni versity of Pennsylvania, Philadelphia, PA). The desired mutations in human 2 subunits (T59A, T59S and T59Y) were created using the QuikChange site-directed mutagenesis kit (Strat agene, La Jolla, CA). The mutated plasmids containing staggered nicks were firstly generated by the thermal cycling reaction with the primers as follows: 5 -ccaatgtctggctggcccaggagtgggaa-3 and 5 -ttcccactcctgggccagccagacattgg-3 for T84A; 5 -ccaatgtctggctgtcccaggagtgggaa-3 and 5 -ttcccactcctgggacagccagacattgg-3 for T84S; 5 -ccaccaatgtctggctgta ccaggagtgggaagatt-3 and 5 -aatcttcccactcctggtacagccagacattggtgg-3 for T84Y. The amplification products were digested with Dpn I endonuclease at 37 oC for 1 hour to eliminate the parental DNA template. Then, the DNAs containing the desired mutation were transformed into XL1-Blue supercompetent cells After transformation, the selected colonies from LB-ampicillin plates were re-incubated to amplify plasmid DNA, which was subsequently purified with QIAprep spin miniprep Kit (Qiagen, Germantown, MD). The desired mutants were finally verified by DNA sequencing (ICBR, University of Florida). 17


In vitro Synthesis of mRNA and Oocyte Expression The subunit mRNAs were prepared from linearized cDNA using the SP6 mMessage mMachine kit (Ambion, Austin, TX). The cDNA coding human 4 subunit was linearized with AseI, and the 2 subunit with PvuII. The mRNA transcripts of human 4 (500 ng/l) and 2 subunits (500 ng/l) were mixed in a ratio of 1:1 before microinjection. Stage V and VI oocytes were surgically harvested from Xenopus laevis anesthetized with ethyl 3-aminobenzoate methanesulfonate salt, then treated with 1.25 mg/ml collagenase (dissolved in calcium-free Barths solution) fo r 2 hours at room temperature to remove the follicular cell layer. Subsequently, the isolated oocytes were maintained in the Barths solution (88 mM NaCl, 1 mM KCl, 0.81 mM MgSO4, 0.41 mM CaCl2, 0.32 mM Ca(NO3)2, 2.38 mM NaHCO3,15 mM HEPES, and 0.01 mg/ml tetracycline hydrochloride, pH 7.1). After one day incubation, each oocyte was injected with 50 nl of the appropriate mRNA mixture by Nanoject II Injector (Drummond, Broomhall, PA). The injected oocytes were incubated at 19 oC for 3 days in a modified Barths soluti on that was renewed daily. Electrophysiology and Data Analysis Electrophysiological recording were performed 3 days after microinjection using twoelectrode voltage clamp. The voltage electrode was filled with 3 M KCl, and current electrode was filled with the solution containing 250 mM CsCl, 250 mM CsF and 100 mM EGTA, pH7.3. Oocytes were placed at room temperature in the recording chamber perfused with frog Ringers solution (115 mM NaCl, 10 mM HEPES, 2.5 mM KCl, 1.8 mM CaCl2, and 1 mM atropine, pH 7.3). Each oocyte was voltage-clamped at a holdi ng potential of -50 mV. The intact experiments were performed using an Axoclamp-2 amplif ier (Axon, Burlingame, CA) and a DigiData 1200 interface (Axon, CA). Each oocyte r eceived two initial control appli cations of ACh, a subsequent drug application, and a terminal control app lication of ACh. Between each drug application 18


oocyte was washed with frog Ringers solution fo r 10 minutes in order to avoid superimposed response. For DH E inhibition assay, DH E was coapplied with 30 M of ACh for 5 seconds. Data were collected using pClamp 8 software (Axon, CA). Peak current responses to the test drug were scaled after base line subtraction using Clampfit 8 software (Molecular Devices). For dose-response curve, data were fitted with a sigmoidal dose response with variable slope: Hillslope XECBottomITopI BottomIY ) (log max max max50101 where X denotes the logarith m of concentration of test compounds; Y denotes the peak current response; ImaxTop = Y value at the top plateau of the curve; ImaxBottom = Y value at the bottom plateau of the curve. Statistical significance was assessed using a two-tailed unpaired t test. 19


CHAPTER 3 RESULTS [3H]-DHSOV Saturation Binding in Rat Brain Membranes (RBM) Previous study using cytisine displacement binding a ssays indicated that DHSOV possesses a higher affinity for the 4 2 receptor than erysovine (Wildeboer, 2005). Here I directly measured the affinity of [3H]-DHOSV binding to rat brain membrane 4 2 nAChRs. As described in the previous chapter, th e nonspecific binding was determined by [3H]-DHSOV binding in the presence of nicotine. In Figure 3-1, specific binding of [3H]-DHSOV, defined as the difference between in the absence and presen ce of nicotine, was saturable over the ligand concentration range 0.05-20 nM. When the [3H]-DHSOV saturation binding assay was initially performed in the narrow concentration range 0.2-10 nM (Figure 3-1 A and B), the binding data was fitted best to the model for one site binding with Kd value of 2 nM. However, when this assay was performed over a wider concentra tion range 0.05-20 nM, the binding data was fitted best to the model for two sites binding (Figure 3-1 C and D). The calculated high affinity and the low affinity Kis for DHSOV binding were 0.6 nM and 7 nM, respectively. Influence of pH on [3H]-DHSOV Binding in Rat Brain Membranes In order to test the hypotheses that ionized DHSOV is the form which avidly binds to 4 2 nAChR, I determined the specific binding of [3H]-DHSOV at pHs 7.4, 8.2 and 9.0. Over the 0.05 to 20 nM concentration range all binding data at the three different pHs were fitted best to the two sites binding model (Table 3-1). The DHSOV high affinity site displayed no significant change at different pH, whereas the DHSOV low affinity site si gnificantly changed at these different pHs. At a lower pH, the compound is in a more ionized form. At a higher pH (> pKa), the compound is a predominantly unionized fo rm. The percent ionization of a basic compound can be calculated from the known pKa according to the Henderson-Hasselbalch equation. Based 20


on the known pKa of other related Erythrina compounds, we predict that the pKa of DHSOV is approximately 8.6. The DHSOV low affinity (Kd2) markedly increased with increasing pH over this range. This suggests that the ionized form of DHSOV has higher affinity for this subtype of nAChR. Displacement of [3H]-DHSOV Binding in Rat Brain Membranes by Agonists and Antagonists In order to further explore the potential of [3H]-DHSOV as the radiolabeled probe for 2containing nAChRs, we tested several co mmon nAChR ligands in cluding agonists and antagonists to displace specific binding of [3H]-DHSOV. By comparing these ligand Ki values with those from the displacement assays using [3H]-cytisine under identical conditions, we found that the Kis of agonists for displacement of [3H]-DHSOV were 3-47-fol d higher than those for displacement [3H]-cytisine. However, there were co mparatively small differences in Kis of antagonists between using antagonist as radioligand and using agoni st as radioligand. Therefore, agonist binding to 4 2 nAChR displays state-dependence. In the above displacement binding assays, 5 nM of [3H]-DHSOV was used. I found that the displacement curves for some common nAChR ligands such as DMPP, RJR-2403, (+)-anatoxin A, and anabaseine were shallow (Hill slope < 0.6). Considering the tw o binding sites model in RBM, the phenomena is possibly due to the balance between the number of the DHSOV high affinity (0.6 nM) and low affinity (7 nM) binding sites in RBMs and the rela tive affinities of the displacing ligand for these two sites. When 1 nM [3H]-DHSOV was used in the above mentioned ligands displacement binding assays, the Hill slop es for these agonists increased as shown in Table 3-3. The occurrence of these steep Hill slop es for agonist displacement of radioligand at this lower concentration is consistent with the occurrence of both the low and high affinity sites for DHSOV in RBM. 21


[3H]-DHSOV Saturation Binding in TSA-201 Cells Expressing 4 2 nAChRs In Figure 3-1, [3H]-DHSOV binding displayed two different affinities sites for rat brain membranes. To make clear which receptor subtype di splays low affinity site or high affinity site for DHSOV, we measured the DHSOV saturation binding in TSA-201 cells that specifically express 4 2 nAChRs. The saturation binding in TSA-201 cel ls was fitted best to the model for one site binding (Figure 3-2). The Kd of DHSOV for 4 2 nACh was approximately 8 nM, which is close to the value for the low affinity binding site in rat brain membranes (7 nM, Figure 3-1). The expression of 4 2 nAChRs in TSA-201 cells will produces two stoichiometries, ( 4)2( 2)3 and ( 4)3( 2)2. They differ in functional pharmacology, desensitization kinetics, unitary conductance and sensitiv ity to chronic exposure to nico tine (Moroni and Bermudez, 2006; Nelson et al., 2003). It is possible that these two 4 2 nAChRs might result in two binding site subpopulations as in the two-site model observe d in RBM. In Figure 3-3, 20 M nicotine was used to treat TSA-201 cells overnight in order to increase the proportion of ( 4)2( 2)3 receptor as in Kuryatov et al. (2005). We found that the Kd of DHSOV did not decrease although the Bmax significantly increased (shown in Figure 3-3). We also determined the saturation binding of DHSOV in TSA cells specially expressing 4 5 2 nAChRs obtained from the Lindstrom lab. The Kd of DHSOV (Figure 3-4) only slightly decreased to 3 nM. Threonine 84 of 2 May Be Involved in DH E Binding In order to better understand Erythrina alkaloid binding to 4 2 nAChR, we constructed the simple molecular model of DHSOV docking in the collaboration with Dr. Alan Katritzkys lab. The model showed that the loop D at the complementary side of Aplysia AChBP has close contact with 3-methoxy group at ring A of the Erythrina alkaloid. In this study, we introduced 22


alanine, serine and tyrosine residues at T84 position, which is located at loop D of the 2 subunit. A hydroxyl group is present in the side chain of threonine, serine or ty rosine, but no alanine. First, ACh dose-response curves for wild-type or mutant 4 2 receptors were determined by Dr. Xing, using my RNAs for expression in the Xenopus oocytes. The EC50 values for wild type 4 2, 4 2T84A, 4 2T84S and 4 2T84Y were 11.2 1.39, 9.0 1.32, 7.6 1.39 and 10.7 1.62 M, respectively (shown in Figure 3-5). Thus, mutation of T84 on the 2 subunit did not significantly change 4 2 receptor activation by ACh ( p > 0.05). It suggested that the hydroxyl group of threonine 84 is not important for ACh binding to 4 2 receptor. Considering of the very similar EC50 value in wild type and mutant 4 2 receptor, the same concentration of ACh (30 M) could be used in the following DH E inhibition response. In Figure 3-6, 30 M of ACh wa s coapplied with different DH E concentrations to measure DH E inhibition responses to wild-type or mutant 4 2 receptors. The response of DH E inhibition for 4 2T84A receptor slightly shifted to the right of the wild type 4 2 response. In contrast, the mutant of 4 2T84S and 4 2T84Y did not significantly change the DH E sensitivity to 4 2 receptor, indicating that DH E sensitivity may be partially attributable to the hydroxyl group in the side chain of threonine, serine or tyrosine. 23


0.0 2.5 5.0 7.5 10.0 12.5 0 10 20 30 40 50 Kd=2 nM [3H]-DHSOV (nM)[3H]-DHSOV Bound (fmol/mg protein) 0 10 20 30 40 50 0 10 20 30 Kd=2 nM [3H]-DHSOV Bound (fmol/mg protein)[3H]-DHSOV Bound/Free (fmol/mg protein/nM) 0 5 10 15 20 25 0 25 50 75 BMAX1 KD1 BMAX2 KD2 19 0.6 57 7[3H]-DHSOV (nM)[3H]-DHSOV Bound (fmol/mg protein) 0 10 20 30 40 50 60 70 0 25 50 75KA B K .15 nM d = 2 0C D d1= 0.6 0.09 nM Kd2= 7 0.2 nM [3H]-DHSOV Bound (fmol/mg protein)[3H]-DHSOV Bound/Free (fmol/mg protein/nM) Kd1 = 0.6 0.09 nM Kd2 = 7 0.2 nM Figure 3-1. [3H]-DHSOV binding in rat brain memb ranes. A, Saturation binding of [3H]-DHSOV at the concentration of 0.2-10 nM by non -regression analysis. Nonspecific binding was determined in the presence of 1 mM (-)-n icotine. B, Scatchard plot of specific [3H]-DHSOV binding shown in A. C. Saturation binding of [3H]-DHSOV at the concentration of 0.05-20 nM. D. Scatchard plot of specific [3H]-DHSOV binding shown in C. The Kd and Bmax values shown were calculated from nonlinear regression analysis. Each mean of Kd was obtained from at least three experiments. Each concentration in an experiment was performed in quadruplicate. 24


Table 3-1. The specific binding of [3H]-DHSOV at pHs 7.4, 8.2 and 9.0 to rat brain membranes. The concentration range of [3H]-DHSOV is from 0.05 to 20 nM. Values are the mean SEM. All data are obtained from three in dependent experiments. The percentage of ionization is calculated using th e Henderson-Hasselbalch Equation ( ][ ][ unionized ionized logpKpH10 a) and pKa=8.6. The abbreviation N.M. is Not Measurable. pH 7.4 8.2 9.0 % ionization 94.1 71.5 28.5 Bmax1 (fmol/mg) 20 2.5 30 6.8 11 1.8 Kd1 (nM) 0.6 0.09 1.1 0.38 1.4 0.49 Bmax2 (fmol/mg) 53 2.9 N.M. N.M. Kd2 (nM) 7.4 0.15 70.3 3.5 >500 25


Table 3-2. Ki values of some common nAChR ligands as determined by displacement of [3H]DHSOV and other radioligands selective for the 4 2 nAChR. 5 nM of [3H]-DHSOV was used in the experiments. The Kd value for [3H]-DHSOV binding to rat brain membranes used in the Cheng-Pr usoff equation to calculate Ki of the displacing ligand was 7 nM (Figure 3-1). The efficacy of a ligand was calculated by normalizing the maximum current response to ligand to the maximum current response to ACh. The ratio is Ki using [3H]-DHSOV as radioligand to Ki using [3H]-cytisine as radioligand. Compound Efficacy (% of ACh) Ki (nM) Radiolabeled Antagonist [3H]-DHSOV Ki (nM) Radiolabeled Agonist [3H]-Cytisine Ratio A-85380 134 a 0.54 0.14 0.05 0.01 a 10.8 Epibatidine 125 b 0.22 0.05 0.04 0.01 f 5.5 Acetylcholine 100 558 130 12 4 g 47 RJR-2403 83 c 190 107 45 9 4.2 Carbachol 78 3275 913 225 33 g 14.5 Nicotine 71 c 29 2 3.3 1.0 g 8.8 Cytisine 32 b 4.3 1.2 0.45 0.15 g 9.6 UB165 30 d 3.5 1.3 0.77 0.08 4.5 Anabaseine 8 e 414 155 75 16 h 5.5 Agonist (+)-anatoxin A 40 d, 8.8 3.2 3.3 0.3 2.7 DH E 0 156 30 140 18 i 1.1 GTS-21 0 621 137 253 37 j 2.5 MLA 0 3670 1100 1583 106 f 2.3 Antagonist d-Tubocurarine 0 34600 6200 N.D. N.D. a Sullivan et al., (1996), b Gopalakrishnan et al., (1996), c Papke et al., (2000), d Sharples et al., (2000), e Kem et al., (1997), f Anderson et al., (1995), g Pabreza et al., (1991), h de Fiebre et al., (1995), i Wildeboer, (2005), j Kem et al., (2004), N.D., Not Determinied. The efficacy of (+)-anatoxin A was measured by [3H]-dopamine release from striatal synaptosomes, normalizing to epibatidine stimulation. 26


Table 3-3. Comparison of Hill slopes and Kis obtained from displacement experiments when two different concentrations of [3H]-DHSOV were tested. The Kd value for 5 nM [3H]DHSOV binding to rat brain membranes used in the Cheng-Prusoff equation to calculate Ki of the displacing ligand was 7 nM. The Kd value for 1 nM [3H]-DHSOV binding used in equation was 0.6 nM. Values are the mean SEM. All data are obtained from three independent experiments. 5 nM of [3H]-DHSOV 1 nM of [3H]-DHSOV Compound nHill slope Ki (nM) nHill slope Ki (nM) DMPP -0.66 0.20 469 127 -0.72 0.08 55 12 RJR-2403 -0.57 0.08 190 107 -0.76 0.08 19 5 (+)-Anatoxin A -0.53 0.02 8.8 3.2 -0.92 0.16 1.98 0.39 Anabaseine -0.46 0.05 414 155 -0.76 0.04 35 1 27


0 5 10 15 20 25 0 100 200 [3H-DHSOV (nM)[3H-DHSOV Bound (fmol/mg protein) Kd = 8 0.4 nM Figure 3-2. [3H]-DHSOV saturation bindi ng in TSA-201 cells at the concentration of 0.05-20 nM. The Kd and Bmax values shown were calculated from nonlinear regression analysis. The means of Kd was obtained from two experiments. Each experiment was performed in quadruplicate. 28


0 5 10 15 20 25 0 1000 2000 3000 [3H-DHSOV], nM[3H]-DHSOV Bound (fmol/mg protein) Kd = 25 4.1 nM Figure 3-3. [3H]-DHSOV saturation bindi ng in TSA-201 cells at the concentration of 0.05-20 nM after 20 M nicotine treatment of TS A-201 cells over night (~16 hours). The Kd and Bmax values were calculated from nonlinea r regression analysis. The means of Kd was obtained from two experiments. Each expe riment was performed in quadruplicate. 29


0 5 10 15 20 25 0 25 50 75 [3H-DHSOV], nM[3H]-DHSOV Bound (fmol/mg protein) Kd = 3 1.5 nM Figure 3-4. [3H]-DHSOV saturation bindi ng in TSA cells expressing 4 5 2 nAChRs at the concentration of 0.05-20 nM. The Kd and Bmax values were calculated from nonlinear regression analyses. The means of Kd was obtained from two experiments. Each experiment was performed in quadruplicate. 30


Wild type 4 2 -7 -6 -5 -4 -3 0 50 100 150 4 2, T84A on 2 4 2, T84S on 2 4 2, T84Y on 2 EC50 4 2, T84A on 2 9.0 M (n=4) 4 2, T84S on 2 7.6 M (n=4) 4 2, T84Y on 2 10.7 M (n=4) Log [ACh], MPercent of Maximal ResponseWild type 4 2 11.2 M (n=4) Figure 3-5. ACh dose-response curves for wild type (solid square), 2T84A (solid triangle), 2T84S (blanked square), and 2T84Y (blanked triangle) 4 2 receptors. Six ACh concentrations (1, 3, 10, 30, 100, 300 M) were tested by voltage clamp recording. Each point shows the averaged value (n 4) normalized to the maximum ACh responses from the same oocytes. 31


-8 -7 -6 -5 -4 -3 -20 0 20 40 60 80 100 120 wild type 4 2 4 2, T84A on 2 4 2, T84S on 2 4 2, T84Y on 2 IC50 4 2, T84A on 2 1.46 M (n 4 2, T84S on 2 0.51 M (n=4) 4 2, T84Y on 2 0.60 M (n=4) Log [DH E], MNormalized Response (%)wild type 4 2 0.65 M (n=5) Figure 3-6. DH E inhibition curves for wild type (solid square), 2T84A (solid circle), 2T84S (blanked circle), and 2T84Y (blanked square) 4 2 receptors. Five ACh concentrations (0.03, 0.1, 1, 10, 100 M) were coapplied with 30 M of ACh. The response of co-application was normalized to the response of the same oocyte to 30 M of ACh alone. Symbols are the average and SEM at least four separate oocytes. 32


CHAPTER 4 DISCUSSION The results of [3H]-DHSOV binding assays (Figure 3-1) indicate that rat brain membranes have at least two binding sites for DHSOV with different affinities (0.6 0.09 nM and 7 0.2 nM). The saturation binding assays with TSA-201 cells (Figure 3-2) provide strong evidence that 4 2 receptor is one of the receptor subtypes in RBMs which contributes to the DHSOV low affinity binding site. It has been found that two stoichiometries of 4 2 receptors are expressed in TSA-201 cells (Moroni and Bermudez, 2006; Nelson et al., 2003). The majority of 4 2 nAChRs expressed in TS A-201 cells are in the ( 4)3( 2)2 form. Both in the oocyte expression system and in the transfected cell line, ( 4)3( 2)2 receptor is less sensitiv e to acetylcholine than is the ( 4)2( 2)3 receptor (Moroni and Bermudez, 2006; Nelson et al., 2003). Besides acetylcholine, it has been found that the antagonist DH E and d-tubocurarine are less potent at ( 4)3( 2)2 receptor than in ( 4)2( 2)3 receptor (Moroni et al., 2006). We originally hypothesized that DHSOV had different affinities for these two stoichiometries of 4 2 receptors, to explain the two binding sites in RBM as shown in Figure 31. Nelson et al. (2003) found that overnight incubation TS A cells with 5 M nicotine increases the proportion of the other stoichiometry, ( 4)2( 2)3 receptor, to about 32% of the total. If the ( 4)2( 2)3 receptor contributes to the DHSOV high affinity binding si te in RBM (Figure 3-1), one would expected to obtain the two binding si tes model after nicotine preincubation with TSA cells membranes. However, the results (Figure 3-3) do not support this hypothesis. Hence, ( 4)2( 2)3 receptor cannot be the receptor in RBM which represents the DHSOV high affinity binding site. Another 4 2 receptor subtype such as 4 5 2 might be the DHSOV high affinity binding site. It has been found that 4 5 2 receptor in the transfected cell line are 46-fold more sensitive 33


to acetylcholine than are ( 4)3( 2)2 receptor (Kuryatov et al., 2008) The studies from this group also show that the 5-null mutation decreases maximal agonist-evoked 86Rb+ efflux without reducing the total number of nAChRs (Brown et al., 2007). Therefore, we hypothesized that 4 5 2 receptor in RBM result s in the DHSOV high binding site. The DHSOV binding to membranes of the 5-transfected TSA-201 cell line was de termined. The result (Figure 3-4) suggests that DHSOV may display a slightly higher affinity for 4 5 2 receptors than 4 2 receptors. Most nAChR agonists and antagonists cont ain protonatable amine or quaternary ammonium (permanently charged) groups, and ther e is considerable evidence that the cationic species is the active form for agonists (Jeng and Cohen, 1980; Zhong et al., 1998). The previous data from our laboratory on pH dependence of GTS-21 binding to 7 nAChR showed that monocationic form of the ligand has the highest a ffinity to nicotinic receptor (Kem et al., 2004). A more recent study of the related 3-(4-aminobenzylidene)-anabasein e found that only the monocationic form binds to AChBP (Talley et al ., 2006). My current data on pH dependence of DHSOV binding is most consistent with its protonated form also having the highest affinity for 4 2 receptors. Interpretation of these experiment s assumes that changes in receptor ionization are not involved. The RBM high a ffinity binding site showed less dependence on pH than the low affinity site, but nevertheless its affinity decreased as the pH increased, as would be expected if the protonated species binds with high er affinity than the unionized species (Table 31). The estimation of the binding properties of this high affinity binding site may be less accurate the Bmax for these sites was roughly 20-30% of the num ber of sites for the high affinity sites. Thus, we cannot exclude the possibility that the high affinity site has a similar pH dependence based on our data. 34


The ionization state of rat nAChR may change at the same time as DHSOV protonation. It is not clear if this contributes to the pH dependence of DHSOV binding. It has been observed that the ACh-induced peak currents of Torpedo nAChR increase with the increasing extracellular pH (5.5-9.0). In contrast, the peak current of mouse muscle nAChR displayed the bell-shaped pH dependence with the peak at pH 7.4 (Li and McNamee, 1992). Modulation of nAChR by pH is apparently dependent on the type of receptor. The influence of pH on 4 2 and 4 5 2 receptor protonation is still unclear. The three-state model for nAChRs defined the resting state, activated state and desensitized state as three main states of nAChRs. Nicotinic agonists stabilize the activated and desensitized states of receptor, while compe titive antagonists stabilize the resting state of receptor. The agonists bind activated state of r eceptor with relatively low affinity and the desensitized state of r eceptor with relatively high affinit y. Interpretation of the results of radioligand displacement assay ar e more informative if the radi olabeled ligand is only binding predominantly to one state for which estimates of compound affinities are desired. Currently, the most commonly used radioligands to measure ligand affinity for 4 2 nAChR are agonists such as [3H]-cytisine or [3H]-nicotine. The displacement binding data (Table 3-2) show that agonist binding to 4 2* nAChR shows state-dependence. The Kis for the agonists are lower when measured with [3H]-cytisine than when measured with [3H]-DHSOV. Therefore, [3H]-DHSOV is a more suitable radioligand for measuring ligand affinity of 4 2 receptor resting state than is an agonist radioligand. To identify the amino acid residues crucial for DHSOV and 4 2 interactions, a series of 4 and 2 subunit mutants will be generated in the future by site-directed mutagenesis and the affinity of DHSOV for these mutated 4 2 receptors will be determined. The first amino acid 35


residue I selected for mutation was threonine 84 on the human 2 subunit. Erythrina alkaloid DH E sensitivity to the mutation of this residue, expressed in oocytes, was measured with the help of Dr. Xing. Effects on DHSOV binding will be tested later. The current results with DH E suggest that threonin e 84 of the human 2 subunit may be involved in DH E binding to 4 2 receptor (Figure 3-6). Further experiments will be done for the confirmation of these results. Although the responsiveness of the three mutants to ACh was not significantly different from that of the wild-type receptor (Figure 3-5), the DH E sensitivity of the threonine 84 to alanine mutant was slightly reduced, par tially consistent with the conc lusions of Harvey and Luetje (1996) that this site is important for DH E binding. They found that changing threonine 59 of rat 2 to lysine decreased DH E sensitivity to 3 2 receptor approximately nine fold. Threonine 59 on rat 2 is homologous with threonine 84 on human 2 subunit. Threonine 84 is one of the residues on loop D of the human 2 subunit. In my study, the replacement of threonine with alanine removes the hydroxyl group in the side chain of threonine resulting in less DH E sensitivity to 4 2 receptor. Serine and tyrosi ne mutants at this position, whose side chains have hydroxyl gr oups, did not show a change in DH E sensitivity. A previous study showed that 3-methoxy gr oup at ring A of two similar Erythrina alkaloid is necessary for binding to the 4 2 receptor with high affinity (Wilde boer, 2005). Based on these results and predictions from the molecular docking model, a H-bond may occur between the alkaloid 3methoxy group and hydroxyl group of the 2 subunit threonine 84 when Erythrina alkaloid binds to 4 2 receptors. Threonine is just one of th e residues which might determ ine DHSOV binding to nicotinic receptor. The residues of subunit, especially residues on lo op C and residues 1-94 (Harvey et al., 1996), could be the more crucial determinan ts of DHSOV binding. AChBP loop C contains 36


Tyr185, the disulfide-linked double cysteine 187188 and Tyr 192. It was predicted that the oxygen carbonyl of d-tubocurarine established a hydrogen bond with th e O-H of Tyr 192 of AChBP (on loop C) using molecu lar modeling and docking (Biss on et al., 2005). Considering that DHSOV has relatively high affinity to the 4 2 receptor and relatively low affinity to the 7 receptor and AChBP, I aligned these sequences with 4 subunit and sugges ted that E224, A227 or I229 was important for DHSOV binding. The mu tation of these residues can be done to confirm which residue on subunit loop C is involved in DHSOV interaction with 4 2 receptor. In a word, this thesis mainly focuses on ra dioligand binding directi on with limited scope. Additional experiments, such as functional assays coupled with mutagenesis and molecular modeling, will be needed to further understand how 4 2 receptor interacts with DHSOV. 37


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BIOGRAPHICAL SKETCH Chao Chen was born in 1978 in Bozhou, China, to Qun Chen and Shuhua Li. Chao attended South-Central Universi ty for Nationalities in Wuhan, China where he received his Bachelor of Science degree in biochemistry. In the fall of 2001, he entered Huazhong University of Science and Technology to begin his gradua te career. His project concentrated on the mechanism of elicitor-mediated taxol biosynthesis in Taxus chinensis suspension culture and he received his Master of Engineering in bioc hemical engineering in July 2004. In 2006, Chao entered the Interdisciplinary Program in Biomedical Science at University of Florida College of Medicine. He joined the laboratory of Dr. Willia m R. Kem in the department of Pharmacology and Therapeutics to study the li gand binding properties of nico tinic acetylcholine receptors. 42