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Cloning and Characterization of a Hyperpolarization-Activated, Cyclic Nucleotide-Gated Cation Channel in Aplysia californica

Permanent Link: http://ufdc.ufl.edu/UFE0021709/00001

Material Information

Title: Cloning and Characterization of a Hyperpolarization-Activated, Cyclic Nucleotide-Gated Cation Channel in Aplysia californica
Physical Description: 1 online resource (181 p.)
Language: english
Creator: Kuzyk, Pavlo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: aplysia, cloning, expression, genome, hcn, mollusk, oocyte
Neuroscience (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: Changes in neuronal excitability alter the frequency of neuronal spiking, trigger and modulate associated behaviors and underlie different pathological states. Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels have been suggested to play an important role in modulating neuronal excitability. Most of the properties of HCN channels were studied in vertebrates. However, because neurons of the vertebrate animals are small and neuronal populations are heterogeneous, it is impossible to study the role of the HCN channels on the level of identified individual neurons, neuronal networks and the behaviors they control. In contrast, the marine mollusk, Aplysia californica, has many large identifiable neurons integrated into several well-studied networks. Here, A. californica has been used as a model animal to determine how the molecular organization of the Aplysia HCN channel (acHCN) influences its properties and how the properties of acHCN determine function of the channel on the level of identified individual neurons and neuronal networks. To characterize the channel, the acHCN transcript was first cloned from the CNS of A. californica. The cloned channel is similar to HCN channels from other organisms, but acHCN differs significantly from other HCN channels in its N-terminal region. The first methionine of acHCN is 28 amino acids downstream of the translation start found in other HCN channels indicating that acHCN may be truncated at its N-terminus. However, the region upstream of the first methionine of acHCN exhibits a weak similarity to other HCN channels implying that it may be important for the channel?s functioning. Thus, the question of whether the cloned channel is functional remained. This question was addressed by expressing acHCN in Xenopus laevis oocytes and studying the biophysical and pharmacological properties of the channel. The expressed channel exhibits all major properties of HCN channels, namely, activation by both hyperpolarization and cyclic nucleotides, permeability to potassium and sodium ions and inhibition by Cs+ and ZD7288. Knowing the properties of acHCN and confirming that ZD7288 is its specific blocker, allowed studying the role of the channel in A. californica neurons. Following localization of the acHCN transcript in the CNS of A. californica, three groups of neurons were studied to characterize the functional role of acHCN, i.e., metacerebral cells (MCC) and also buccal motoneuron B3 and pedal locomotory neuron P4, which are part of feeding and locomotory networks, respectively. It was demonstrated that acHCN controls the spiking frequency of these neurons. Together with the fact that spiking of B3 and P4 neurons directly correlates with the contraction of buccal and pedal muscles, respectively (Church and Lloyd, 1994; Hening et al., 1979), this implies a role of acHCN in coordinating feeding and locomotion in A. californica.
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 Pavlo Kuzyk.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Moroz, Leonid L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021709:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021709/00001

Material Information

Title: Cloning and Characterization of a Hyperpolarization-Activated, Cyclic Nucleotide-Gated Cation Channel in Aplysia californica
Physical Description: 1 online resource (181 p.)
Language: english
Creator: Kuzyk, Pavlo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: aplysia, cloning, expression, genome, hcn, mollusk, oocyte
Neuroscience (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: Changes in neuronal excitability alter the frequency of neuronal spiking, trigger and modulate associated behaviors and underlie different pathological states. Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels have been suggested to play an important role in modulating neuronal excitability. Most of the properties of HCN channels were studied in vertebrates. However, because neurons of the vertebrate animals are small and neuronal populations are heterogeneous, it is impossible to study the role of the HCN channels on the level of identified individual neurons, neuronal networks and the behaviors they control. In contrast, the marine mollusk, Aplysia californica, has many large identifiable neurons integrated into several well-studied networks. Here, A. californica has been used as a model animal to determine how the molecular organization of the Aplysia HCN channel (acHCN) influences its properties and how the properties of acHCN determine function of the channel on the level of identified individual neurons and neuronal networks. To characterize the channel, the acHCN transcript was first cloned from the CNS of A. californica. The cloned channel is similar to HCN channels from other organisms, but acHCN differs significantly from other HCN channels in its N-terminal region. The first methionine of acHCN is 28 amino acids downstream of the translation start found in other HCN channels indicating that acHCN may be truncated at its N-terminus. However, the region upstream of the first methionine of acHCN exhibits a weak similarity to other HCN channels implying that it may be important for the channel?s functioning. Thus, the question of whether the cloned channel is functional remained. This question was addressed by expressing acHCN in Xenopus laevis oocytes and studying the biophysical and pharmacological properties of the channel. The expressed channel exhibits all major properties of HCN channels, namely, activation by both hyperpolarization and cyclic nucleotides, permeability to potassium and sodium ions and inhibition by Cs+ and ZD7288. Knowing the properties of acHCN and confirming that ZD7288 is its specific blocker, allowed studying the role of the channel in A. californica neurons. Following localization of the acHCN transcript in the CNS of A. californica, three groups of neurons were studied to characterize the functional role of acHCN, i.e., metacerebral cells (MCC) and also buccal motoneuron B3 and pedal locomotory neuron P4, which are part of feeding and locomotory networks, respectively. It was demonstrated that acHCN controls the spiking frequency of these neurons. Together with the fact that spiking of B3 and P4 neurons directly correlates with the contraction of buccal and pedal muscles, respectively (Church and Lloyd, 1994; Hening et al., 1979), this implies a role of acHCN in coordinating feeding and locomotion in A. californica.
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 Pavlo Kuzyk.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Moroz, Leonid L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021709:00001


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1 CLONING AND CHARACTERIZATION OF A HYPERPOLARIZATION-ACTIVATED, CYCLIC NUCLEOTIDE-GATED CATION CHANNEL IN APLYSIA CALIFORNICA By PAVLO KUZYK DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Pavlo Kuzyk

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3 To my parents, Galyna and Mykhaylo Kuzyk

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5 ACKNOWLEDGMENTS I would like to thank my mentor, Leonid Moroz, and members of my supervisory committee, Peter Anderson, Barbara Battelle, Ge rry Shaw and Weihong Tan for their constant attention during the years of my graduate study. I am also grat eful to all the people who helped me with different aspects of my work. David Pr ice, Yuri Panchin and Sergey Kalachikov gave me much useful advice about cloning of acHCN. David Price also provided a plasmid for expression of acHCN in Xenopus oocytes, Sergey Kalachikov helped me develop a Northern blot protocol, and Yuri Panchin provi ded a serotonin transporter clone Becky Price and Lala Popova taught me how to record from Xenopus oocytes. Jocelyn Tulsian performed surgery and removed oocytes from the frogs. Dmitri Boudko kindly allowe d me to use his rig for electrophysiological recordings from oocytes. Yuri Bobkov answered numerous questions I had about the properties of HCN channels and helped me to analyze electrophysiological data. Elena Bobkova provided significant technical support. Sami Jezzi ni helped me with recording from Aplysia neurons and with different scientific problems. Terri Walters showed me how to do surgery on Aplysia Naila Alieva taught me how to do in situ hybridization. Jim Netherton a nd members of SciComm class read and commented on my manuscripts and help ed to prepare presentations. Also I thank Christelle Bouchard, Lala Popova and Kristen Me rritt for their moral support, especially during hard periods of my graduate study.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 LIST OF ABBREVIATIONS........................................................................................................12 ABSTRACT....................................................................................................................... ............14 CHAPTER 1 INTRODUCTION..................................................................................................................16 1.1 HCN Channels and Their Ro le in Neuronal Excitability................................................16 1.2 Molecular Organization of HCN Channels.....................................................................18 1.3 Aplysia californica as a Model to Study HCN Channels................................................19 2 CLONING AND MOLECULAR CHARACTERIZATION OF THE acHCN CHANNEL........................................................................................................................ .....28 2.1 Introduction.............................................................................................................. ........28 2.2 Materials and Methods....................................................................................................29 2.2.1 Materials and Reagents...........................................................................................29 2.2.2 Cloning and Sequence Analysis............................................................................29 2.2.3 Determination of the Genomic Organization of acHCN.......................................31 2.2.4 Northern Blot.........................................................................................................31 2.2.4.1 RNA isolation and blotting.........................................................................31 2.2.4.2 Chemiluminescent detection.......................................................................32 2.2.4.3 Radioactive detection..................................................................................33 2.3 Results................................................................................................................... ...........33 2.3.1 Identity and Molecular Organization of acHCN...................................................33 2.3.2 Duplication of a Coding Re gion at the 3-UTR of acHCN...................................36 2.3.3 Genomic Organization and Possi ble Splicing Variants of acHCN.......................37 2.3.4 Northern Blot.........................................................................................................38 2.4 Discussion................................................................................................................ ........40 2.4.1 Identity and Molecular Organization of acHCN...................................................40 2.4.2 HCN Channels in Invertebrates.............................................................................41 2.4.3 Cyclic Nucleotide-Binding Channe ls in Non-Animal Eukaryotes........................42 2.4.4 Cyclic Nucleotide-Binding Potassium Channels in Prokaryota............................43 2.4.5 Origin and Potential Evolutio n of HCN and Related Channels............................44 2.4.6 Duplication of a Coding Region at the 3-UTR of acHCN and Genomic Organization of the Channel..................................................................................45 2.4.7 Possible Splice Variants of acHCN.......................................................................46 2.4.8 Verification of Channel Length.............................................................................46

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6 2.4.5 Summary................................................................................................................47 3 BIOPHYSICAL AND PHARMACOLOGICA L CHARACTERIZATION OF acHCN......65 3.1 Introduction.............................................................................................................. ........65 3.2 Methods................................................................................................................... ........66 3.2.1 acHCN RNA Synthesis.........................................................................................66 3.2.2 Expression in the Xenopus Oocytes......................................................................67 3.2.3 Voltage-Clamp Recording of the Whole-Oocyte Responses................................68 3.2.4 Data Analysis.........................................................................................................70 3.3 Results................................................................................................................... ...........70 3.3.1 Voltage Dependence and Kinetics.........................................................................70 3.3.2 Ion Permeability of acHCN...................................................................................71 3.3.3 Activation of acHCN by Cyclic Nucleotides........................................................72 3.3.4 Inhibition of acHCN by ZD7288 and Cs+.............................................................73 3.4 Discussion................................................................................................................ ........74 3.4.1. Voltage Dependence and Kinetics.........................................................................74 3.4.2 Ion Permeability of the acHCN Channel...............................................................77 3.4.3 Activation of the acHCN Ch annel by Cyclic Nucleotides....................................77 3.4.3 Inhibition of the acHCN Channel by Cs+ and ZD7288.........................................78 3.4.5 Summary................................................................................................................79 4 LOCALIZATION OF acHCN IN APLYSIA CALIFORNICA ................................................96 4.1 Introduction.............................................................................................................. ........96 4.2 Methods................................................................................................................... ........98 4.2.1 Animals................................................................................................................. .98 4.2.2 Surgery................................................................................................................. .98 4.2.3 Whole-Mount i n situ Hybridization Protocol........................................................98 4.2.4 Imaging................................................................................................................100 4.2.5 Densitometry and Cell Counts.............................................................................100 4.3 Results................................................................................................................... .........100 4.3.1 Localization of acHCN RNA in A. californica ...................................................100 4.3.2 Down-Regulation of the acHCN Transcript Following Nerve Injury.................101 4.4 Discussion................................................................................................................ ......103 4.4.1 Localization of the acHCN RNA in A. californica .............................................103 4.4.2 Down-Regulation of the acHCN Transcript Following Nerve Injury.................105 4.4.3 Summary..............................................................................................................106 5 CHARACTERIZATION OF THE FUNCTIONAL ROLE OF acHCN..............................117 5.1 Introduction.............................................................................................................. ......117 5.2 Methods................................................................................................................... ......118 5.2.1 Electrophysiological Recording from A. californica Neurons............................118 5.2.2 Data Analysis.......................................................................................................119 5.3 Results................................................................................................................... .........119 5.3.1 Specificity of ZD7288 in A. californica ..............................................................119 5.3.2 Effect of ZD7288 on the Spiking Frequency of Individual Neurons Expressing acHCN...............................................................................................120

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7 5.3.3 Effect of ZD7288 on Systemic Level..................................................................121 5.4 Discussion................................................................................................................ ......121 6 SUMMARY AND FUTURE WORK..................................................................................132 APPENDIX A SEQUENCES OF THE HCN CHANNELS USED TO CONSTRUCT PHYLOGENETIC TREE AND THEIR ALIGNMENT......................................................136 B SEQUENCES OF THE CNBD-CONTAINING POTASSIUM AND CATION CHANNELS USED TO CONSTRUC T PHYLOGENETIC TREE AND THEIR ALIGNMENT...................................................................................................................... .143 C RECORDING OF THE PHYSALIA VOL TAGE-GATED POTASSIUM CHANNEL EXPRESSED IN OOCYTES...............................................................................................163 D CLONING AND LOCALIZATION OF A CYCLIC NUCLEOTIDE-GATED CHANNEL IN APLYSIA CALIFORNICA ...........................................................................164 CNG Cloning and Pr obe Synthesis.......................................................................................164 Localization of the CNG RNA in the CNS of A. californica ...............................................164 Discussion..................................................................................................................... ........165 LIST OF REFERENCES.............................................................................................................168 BIOGRAPHICAL SKETCH.......................................................................................................181

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8 LIST OF TABLES Table page 1-1. HCN channels in different animal phyla...............................................................................26 2-1. Sequence and position of exonintron junctions of acHCN gene..........................................62 2-2. Identities of nucleotides in th e positions 1-4 shown in Figure 2-9........................................63 2-3. Comparison of properties of HCN cha nnels and other channels containing a CNBD..........64 3-1. Characteristics and properties of HCN channels cloned from different organisms..............95 4-1. Expression of the ac HCN mRNA in the CNS of A. californica ..........................................116 6-1. Comparison of acHCN and other HCN channels................................................................135

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9 LIST OF FIGURES Figure page 1-1. Biophysical properties of HCN channels..............................................................................21 1-2. Schematic diagram of neuronal spiking in control conditions (black) and in the presence of cyclic nucleotides (red)......................................................................................22 1-3. Two-dimensional model of an HCN channel........................................................................23 1-4. Sea slug Aplysia californica ..................................................................................................24 1-5. Abdominal ganglion of A. californica ...................................................................................25 2-1. The longest cDNA sequence and pred icted amino acid sequence of acHCN.......................48 2-2. Alignment of HCN protei ns from different organisms.........................................................49 2-3. Alignment of the voltage sensors from the HCN channels of different organisms...............50 2-4. Alignment of the pore regions from th e HCN channels of different organisms...................51 2-5. Phylogenetic tree of HCN channels.......................................................................................52 2-6. Experiments showing the existence of a duplicate sequence at the 3-end of acHCN..........53 2-7. Genomic organization of acHCN..........................................................................................54 2-8. Number of exons in HCN ge nes in different animal phyla...................................................55 2-9. Sequence of exon 9....................................................................................................... .........56 2-10. Comparison of Northern blots using probes to acHCN and FMRFamide (control)...........57 2-11. Phylogenetic tree of potassium and ca tion channels containi ng a cyclic nucleotidebinding domain................................................................................................................. .58 2-12. Phylogenetic trees showing the number of HCN genes in different phyla of the animal kingdom........................................................................................................................ .....60 2-13. Two forms of the acHCN channel that may be synthesized................................................61 3-1. Schematic diagram of the cons tructs used to produce acHCN RNA....................................81 3-2. Schematic diagram of a two-electrode voltage clamp oocyt e recording set-up....................82 3-3. Determination of the voltage dependence of the acHCN channel.........................................83 3-4. Voltage dependence of the acHCN channel activation.........................................................84

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10 3-5. Determination of a reversal potential of the acHCN channel................................................85 3-6. Dependence of the reversal potential of the acHCN channel on extracellular potassium concentration.................................................................................................................. .......86 3-7. Dependence of the Ih amplitude on extracellular potassium concentration...........................87 3-8. Activation of the acHCN channel by cyclic AMP................................................................88 3-9. Activation of the acHCN channel by cyclic GMP................................................................89 3-10. Concentration dependence of acHCN activation by 8-Br-cGMP.......................................90 3-11. Concentration dependence of acHCN activation by 8-Br-cAMP.......................................91 3-12. Inhibition of the acHCN channel by low concentrations of Cs+.........................................92 3-13. Inhibition of acHCN by ZD7288.........................................................................................93 3-14. Concentration dependence of th e acHCN channel inhibition by ZD7288..........................94 4-1. Surgery design for pedal nerve crush..................................................................................107 4-2. Schematic diagram of in situ hybridization.........................................................................108 4-3. Expression of the acHCN chan nel transcript in the CNS of A. californica as determined by in situ hybridization.......................................................................................................109 4-4. Schematic diagram of the expr ession of the acHCN transcript...........................................110 4-5. Expression of acH CN in the aorta of A. californica as determined by in situ hybridization.................................................................................................................. .....111 4-6. acHCN mRNA expression is de creased following nerve injury.........................................112 4-7. Expression of tryptophan hydroxylase mRNA is decreased following nerve injury in pedal ganglia both ipsiand cont ralateral to the nerve crush..............................................113 4-8. Expression of serotonin transporter mRNA in the A. californica CNS as determined by in situ hybridization............................................................................................................114 4-9. Expression of serotonin transporter mRNA is decreased following nerve injury...............115 5-1. Determination of the sp ecificity of ZD7288 for acHCN.....................................................125 5-2. Effect of ZD7288 on spiki ng of the metacerebral cells.......................................................126 5-3. Effect of ZD7288 on spiking of the s ynaptically-isolated metacerebral cells.....................127 5-4. Comparison of the effect of ZD7288 on spiking of synaptically-coupled and synaptically-isolated MCCs................................................................................................128

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11 5-5. Effect of ZD7288 on spiking of B3 neuron.........................................................................129 5-6. Effect of ZD7288 on spiking of the pedal locomotory neuron P4......................................130 5-7. Effect of ZD7288 on the rhythmic ac tivity of two unidentified buccal neurons expressing acHCN transcript..............................................................................................131 A-1. Alignment of HCN proteins used for construction of the phylogenetic tree......................142 B-1. Alignment of CNBD-containing potassium and cation channels used for construction of the phylogenetic tree.......................................................................................................162 C-1. Recording of the Physalia voltage-gated potassium channel expressed in oocytes...........163 D-1. Expression of an A. californica CNG channel transcript as determined by in situ hybridization.................................................................................................................. .....167

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12 LIST OF ABBREVIATIONS 5-HT 5-hydroxytryptamine (serotonin) Aa amino acids acHCN hyperpolarization-activated, cyclic nucleotide-gated cation channel cloned from Aplysia californica AP action potential AS Aplysia saline cAMP cyclic adenosine monophosphate cGMP cyclic guanosine monophosphate cNMPs cyclic nucleotides CAP catabolite activator protein CNBD cyclic-nucleotide-binding domain CNG cyclic nucleotide-gated channel CNGC (plant) cyclic nucle otide-gated cation channel CNS central nervous system DIG dioxigenin DRG dorsal root ganglion Er reversal potential ERG ether-a-go-go-related channel EST expressed sequence tag HCN hyperpolarization-activated, cycl ic nucleotide-gat ed cation channel Hi-Di high divalent cation solution Ih HCN-mediated current Ka concentration required for half-maximal activation

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13 Ki concentration required for half-maximal inhibition LTH long-term hyperexcitability LTP long-term plasticity MCC metacerebral cells MN motoneuron Nt nucleotide ORF open reading frame PBS phosphate buffer solution PCR polymerase chain reaction PIP2 phosphatidylinositol-4,5-bisphosphate PKA cAMP-dependent protein kinase PKG cGMP-dependent protein kinase PTW 0.1% Tween 20 in PBS RACE rapid amplification of cDNA ends SN sensory neuron activation constant TEA HCl TEA HCl TM transmembrane domain UTR untranslated region V1/2 voltage of the half-maximal activation

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14 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CLONING AND CHARACTERIZATION OF A HYPERPOLARIZATION-ACTIVATED, CYCLIC NUCLEOTIDE-GATED CATION CHANNEL IN APLYSIA CALIFORNICA By Pavlo Kuzyk December 2007 Chair: Leonid L. Moroz Major: Medical Sciences Neuroscience Changes in neuronal excitability alter the frequency of neuronal spiking, trigger and modulate associated behaviors and underlie diffe rent pathological stat es. Hyperpolarizationactivated, cyclic nucleotide-gated cation (HCN) channels have been suggested to play an important role in modulating neur onal excitability. Most of the pr operties of HCN channels were studied in vertebrates. However, because neur ons of the vertebrate animals are small and neuronal populations are heterogeneous, it is impo ssible to study the role of the HCN channels on the level of identified individual neurons, neur onal networks and the behaviors they control. In contrast, the marine mollusk, Aplysia californica has many large identifiable neurons integrated into several well-studied networks. Here, A. californica has been used as a model animal to determine how the molecular organization of the Aplysia HCN channel (acHCN) influences its properties and how the properties of acHCN determ ine function of the channel on the level of identified individual neurons and neuronal networks. To characterize the channel, the acHCN tran script was first cloned from the CNS of A. californica The cloned channel is similar to HCN ch annels from other organisms, but acHCN differs significantly from other HCN channels in its N-terminal region. The first methionine of acHCN is 28 amino acids downstream of the tr anslation start found in other HCN channels indicating that acHCN may be tr uncated at its N-terminus. Howe ver, the region upstream of the

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15 first methionine of acHCN exhibits a weak sim ilarity to other HCN channels implying that it may be important for the channels functioni ng. Thus, the question of whether the cloned channel is functional remained. This ques tion was addressed by expressing acHCN in Xenopus laevis oocytes and studying the biophysical and pha rmacological properties of the channel. The expressed channel exhibits all major prop erties of HCN channels, namely, activation by both hyperpolarization and cyclic nucleotides, permeability to potassium and sodium ions and inhibition by Cs+ and ZD7288. Knowing the properties of acH CN and confirming that ZD7 288 is its specific blocker, allowed studying the role of the channel in A. californica neurons. Following localization of the acHCN transcript in the CNS of A. californica three groups of neurons were studied to characterize the functional role of acHCN, i.e., metacerebral cells (MCC) and also buccal motoneuron B3 and pedal locomotory neuron P4, which are part of feeding and locomotory networks, respectively. It was demo nstrated that acHCN controls the spiking frequency of these neurons. Together with the fact that spiking of B3 and P4 neurons direc tly correlates with the contraction of buccal and pedal muscles, resp ectively (Church and Lloyd, 1994; Hening et al., 1979), this implies a role of acHCN in coordinating feeding and locomotion in A. californica

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16 CHAPTER 1 INTRODUCTION 1.1 HCN Channels and Their Role in Neuronal Excitability Excitability is an important feature of neurons that allows them to process and transmit information. Changes in neuronal ex citability alter the frequency of neuronal spiking, and trigger and modulate the activity of neuronal networks and associated behaviors, e.g., locomotion (Ahlman et al., 1971), reproduction (Conn and K aczmarek, 1989) and aggression (Keele, 2005). Changes in neuronal excitability also underl ie long-term potentiation (LTP) and long-term hyperexcitability (LTH), major pr ocesses of synaptic and non-s ynaptic plasticity, respectively (Kandel, 1976; Weragoda et al., 2004). In the 1970s, Kandel and his colleagues showed that cyclic nucleotides, particularly cyclic adenosine monophosphate (cAMP) and cyclic gu anosine monophosphate (cGMP) contribute to neuronal excitability (Brunelli et al., 1976; Arancio et al., 19 95; Lewin and Walters, 1999). It was demonstrated that cAMP and cGMP depol arize neurons and thus increase neuronal excitability through activation of cAMPand cGMP-dependent protein kinases (PKA and PKG, respectively) that phosphorylate potassium cha nnels, thus reducing their currents (Klein and Kandel, 1980; Siegelbaum et al., 1982). In pa rallel, cAMP and cGMP increase neuronal excitability by recruiti ng different transcription factors that activate expression of genes responsible for the growth of new synaptic conne ctions (Schacher et al., 1988; Dash et al., 1990). However, in addition to the well-studied role of the cyclic nucleotide s in controlling neuronal excitability through these indirect pathways cAMP and cGMP can also regulate neuronal excitability directly by binding to cyclic nucleotide-gated cha nnels, particularly hyperpolarization-activated, cyclic nucleotide-gated (HCN) cation channels (Brown et al., 1979). HCN channels were first found in the pacemaker cells of the rabbit sino-atrial node (Noma and Irisawa, 1976) where they control cardiac rhythmicity (Brown et al., 1979). Shortly

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17 afterward, these channels were also found in the nervous system of guinea pig (Halliwell and Adams, 1982) and shown to control the s pontaneous activity of neurons (Brown and DiFrancesco, 1980; DiFrancesco, 1981; Pape, 1996) and synchronization of the low frequency oscillations in different areas of the brain (Maccaferri and McBain, 1996; Bal and McCormick, 1997; Luthi et al., 1998). In addi tion to controlling the spontane ous activity of different cells, HCN channels also play a role in LTP (Beaumo nt and Zucker, 2000) and different pathological processes, such as neuropathic pain (Chapl an et al., 2003), epilepsy (Chen et al., 2001, 2002), seizure and cardiac ischemia (Robinson and Siegelbaum, 2003) (see Table 1-1). All of the above listed functions are based on the increase of neuronal excitability linked to HCN channels. The ability to increase neuronal excitability, in turn, is determined by the biophysical properties of the channels. HCN ch annels are activated by hyperpolarization, typically negative to -60 mV and ge nerate a slow inward current (Ih) (Figure 1-1A). This current depolarizes cells towards the reversal potential of the channels, which is between -15 and -40 mV. Thus, the resting potential of HCN-expre ssing cells is significantly more positive than a typical resting potential of around -60 mV. Therefore, the HC N-expressing cells may need smaller excitatory synaptic inputs to reach a thre shold for firing an action potential compared to cells that do not express these channels, i.e., the excitability of the HCN-expressing cells can be higher. In electrically-active cells, low-thre shold sodium and calcium channels can act synergistically with HCN channels and further depolarize the cells until they fire an action potential. In addition to hyperpolarization, HCN channels are also activated by cyclic nucleotides. Cyclic AMP and, to a lesser degree, cGMP direc tly bind to HCN channels and shift the voltage dependence of the channel gating to more depolar ized values (DiFrancesco and Tortora, 1991; Ludwig et al., 1998) (Figure 1-1B). Thus, in the presence of cyclic nucleotides, HCN channels

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18 activate earlier during the after-hyperpolarizatio n following an action potential, more quickly depolarize cells towards firing of the next action potential and, ther efore, increase the frequency of neuronal spiking (Figure 1-2). Concentrations of cyclic nucleotides can be greater in neurons undergoing LTP (Cedar et al., 1972), as well as extracellularly, in the cerebros pinal fluid and different regions of the brain following seizure (Myllyla et al., 1975; Ferrendelli et al., 1980) and nerve injury (Ruis-Morales and Vara-Thorbeck, 1986; Siegan et al., 1996). Thus, during these states HCN channels are expected to be activated and to increase neur onal excitability. Also, during seizure and cardiac ischemia HCN channels may be activated by elevated extracellular K+ (Robinson and Siegelbaum, 2003) because an increase in extracellular K+ strongly increases the amplitude of Ih (Figure 1-1C). 1.2 Molecular Organization of HCN Channels The properties of HCN channels, i.e., activa tion by hyperpolarization, cyclic nucleotides and elevated extracellular K+, are determined by the molecula r organization of the channels. HCN channels belong to a superfamily of volta ge-gated potassium channels (Pongs, 1992) and as such have six transmembrane domains (TM) a voltage sensor in TM4 and a pore with a potassium selectivity filter (Figure 1-3). Similar to classic voltage-gated potassium channels, the voltage sensor of HCN channels contains posi tively-charged residues, arginines or lysines, separated from each other by tw o neutral residues (Larsson et al., 1996). However, unlike other potassium channels, which are activated by de polarization, HCN channels are activated by hyperpolarization. This reversed po larity of activation may be de termined by differences in the TM4-TM5 linker and C-terminal region that coup le channel activation and gating (Prole and Yellen, 2006).

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19 The unusual reversal potential of the channels is determined by their permeability to both K+ and Na+. This mixed ionic permeability results from modifications of the potassium selectivity motif TXXTXGYG (Heginbotham et al., 1994) in HCN channels. Whereas the GYG sequence is preserved, the first th reonine residue is altered to histidine and the second threonine residue is changed to either se rine or cysteine. A conserved aspartate, which follows the GYG motif in most potassium channels, is altered in HCN channels to arginine, lysine, glutamine, alanine, serine or methionine. Another difference between HCN and classic voltage-gated potassium channels is the presence of a cyclic nucleotide-binding domain (CNBD) at the C-terminus of HCN channels. As described earlier, all of the changes in HC N channels compared to classic voltage-gated potassium channels result in the ability of HCN channels to regulate ne uronal excitability. 1.3 Aplysia californica as a Model to Study HCN Channels The molecular organization and most of the described properties of HCN channels were studied in vertebrates. However, there are limite d reports about the role of these channels in network functions or systemic function of the br ain. Indeed, neurons of vertebrates are small and neuronal populations are heteroge neous; thus, it is very difficult to study how the activity of the HCN channels in identified indi vidual neurons influences the ex citability and spiking of these neurons, and the activity of the neuronal networ ks and behaviors they control. The CNSs of invertebrate animals have a much simpler organi zation, with smaller numbers of cells organized into well-defined neuronal circui ts. Nevertheless, the majority of neurons in most invertebrate species are also relatively small and difficult to study. In contrast, a marine mollusk, Aplysia californica (Figure 1-4) has many large (up to 1 mm in diameter) id entifiable neurons (Figure 15) that can be easily isolated and used fo r cDNA library construc tion, electrophysiological

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20 recordings, injection, and biochemical analysis which is a difficult to impossible task in vertebrate and most invertebrate syst ems (Kandel, 1976; Moroz et al., 2006). Many neuronal networks controlling identifiab le behaviors have been extensively studied in A. californica including memory-forming (Kandel, 1976, 2001), feeding (Kandel, 1976; Jahan-Parwar and Fredman, 1983; Cropper et al., 2004), defensive (Kupfermann et al., 1970; Castellucci et al., 1970; Kandel, 1976; Croll, 2003) and locomotory networks (Jahan-Parwar and Fredman, 1978; Hening et al., 1979 ; Fredman and Jahan-Parwar, 1983). For example, it is known which pedal neurons control contr action of different parts of the foot (Hening et al., 1979) and which neurons in buccal and cerebral ganglia contro l different aspects of feeding behavior, i.e., protraction and retraction of buccal mass muscles (Jahan-Parwar and Fredman, 1983; Cropper et al., 2004). Locating a transcript of interest in the cells of a cert ain network allows predictions about the role the channel play s in the animals behavior. Subsequent electrophysiological characterization of the channel in these cells provides a means for testing these predictions. Thus, in A. californica it is possible to determine the role of the HCN channel on the level of an individual cell, neuronal networ k and its associated behavior.

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21 Figure 1-1. Biophysical propertie s of HCN channels. A) Determination of the voltage dependence of HCN channel activation. Human HCN1 channel was expressed in human embryonic kidney 293 cells; the cells were voltage-clamped from a holding potential of -40 mV to vari ous potentials ranging from -140 mV to 0 mV, in -10 mV increments and then to -140 mV. Following th e hyperpolarizing step from -40 mV to potentials negative to -70 mV, slow in ward non-inactivating currents developed. Shown in red is the current trace generate d upon stepping from the holding potential of -40 mV to -140 mV. B) Voltage depe ndence of HCN channel activation in the absence of cyclic nucleotides (open circles) or after intrac ellular perfusion with 1 mM cAMP (filled circles) or 1 mM cGMP (tri angles). Cyclic nucleotides shifted the voltage activation of the HCN channel in a depolarizing direction. C) Dependence of Ih amplitude on extracellula r potassium concentration. I/V relationship for the fully activated channel was obtained by plotting the tail-current amplitudes measured after stepping from the holding potential of 40 mV to -140 mV for 1.5 sec and then to more depolarized voltages ranging from 100 to +40 mV in 10 mV steps. I/V relationships were determined at 5.4 mM (filled circles) and 30 mM (open circles) extracellular K+, respectively. Current amplitudes were much larger at 30 mM extracellular K+. Reprinted by permission from Macmillan Publishers Ltd: Nature Ludwig et al., 1998. A B C A B C

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22 Figure 1-2. Schematic diagram of neuronal spik ing in control conditions (black) and in the presence of cyclic nucleotides (red). The potentials of Ih activation are shown by black and red arrows respec tively. In the presence of cyclic nucleotides, HCN channels activate earlier dur ing the after-hyperpolariza tion following an action potential, more quickly depolarize cells to wards firing of the ne xt action potential, and therefore increase frequency of neuronal spiking.

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23 Figure 1-3. Two-dimensional model of an HCN channel. HCN channels have six transmembrane domains (TM1-TM6); an i on pore (P) with a modified potassium selectivity filter; eight positively charged re sidues in TM4 (+) and a cyclic nucleotidebinding domain (CNBD) at the C-terminus.

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24 Figure 1-4. Sea slug Aplysia californica A. californica has characteristic features of the molluscan body plan: head with tentacles, rhinophores (chemosensitive organs) and eyes, foot and mantle cavity containing gills. Head Tentacle Rhinophore Foot Tail Gill Siphon Head Tentacle Rhinophore Foot Tail Gill Siphon

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25 Figure 1-5. Abdominal ganglion of A. californica The abdominal ganglion has many large identifiable neurons with known functions, e.g., L7, which controls gill withdrawal and is a part of a defensive network (K upfermann et al., 1971), neurosecretory neurons R3-R13 (Gainer and Wollberg, 1974), R2, which controls mucus production (Rayport et al., 1983), R15, which is responsib le for respiratory pumping (Alevizos et al., 1991) and bag cluster (BC) cells which produce egg-laying hormone (Kupfermann and Kandel, 1970). Scale bar = 500 m. R2 R3R13 L7 BC R15 R2 R3R13 L7 BC R15 R2 R3R13 L7 BC R15 R2 R3R13 L7 BC R15

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26Table 1-1. HCN channels in different animal phyla. Taxon/species Channel name Accession number Number of exons Function and reference HCN1 gi:32698746 8 HCN2 gi:30580783 8 HCN3 gi:38327037 8 HCN4 gi:4885407 8 Epilepsy (Bender et al., 2003), heart contraction (Vaccari et al., 1999) HCN1 gi:16758108 8 HCN2 gi:50878267 8 HCN3 gi:29840773 8 HCN4 gi:29840771 8 Synchronization in the brain (M accaferri and McBain, 1996), LTH following nerve injury (Chaplan et al., 2003), epilepsy (Chen et al, 2001), hippocampal development (Strata et al., 1997), response to sour stimuli (Stevens et al., 2001), heart activity (Shi et al., 1999) Deuterostomes Phylum Chordata Class Mammalia* Homo sapiens Rattus norvegicus Class Aves Gallus gallus HCN2 gi:86129554 12 Sound source localization (Yamada et al., 2005), generation of the temporal activity patterns in auditory thalamic relay nucleus (Strohmann et al., 1994) Class Amphibia Ambystoma tigrinum HCN Not cloned ? Adaptation to overly intensive stimuli in rod and cone photoreceptors (Bader et al., 1979; Hestrin, 1987) Class Actinopterygii Oncorhynchus mykiss HCN1 gi:33312349 ? May impact release of transmitter from hair cells of the saccule (Cho et al., 2003) Danio rerio HCN1 gi:94732421 8 Contributes to pacemaking in the heart (Baker et al., 1997) SPIH gi:3242324 9 Phylum Echinodermata Class Echinoidea Strongylocentrotus purpuratus SpHCN2 gi:69146514 11 Flagellar beating (Gauss et al., 1998; Galindo et al., 2005) Amih gi:52631748 13 May play a sensory role (Gisselmann et al., 2003) Splice form A1B1C1 gi:24653608 13 May play a sensory role (Marx et al., 1999-2000) Phylum Arthropoda Class Insecta Apis mellifera Drosophila melanogaster Class Crustacea Panulirus argus PAIH gi:33355925 ? Stabilizes membrane polarization and excitability in varying external conditions in stretch receptor ne urons (Edman and Grampp, 1991), may participate in olfactory tran sduction (Gisselmann et al., 2005b), modifies firing activity of stom atogastric ganglion neurons (Zhang et al., 2003)

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27Table 1-1. Continued Phylum Mollusca Class Gastropoda Hermissenda crassicornis HCN Not cloned ? Dark adaptation in in B-type photoreceptors (Yamoah et al., 1998) Lymnaea stagnalis HCN Not cloned ? Modulates activity of central pattern generator underlying rhythmic ingestion movements (Straub and Benjamin, 2001) Phylum Annelida Class Clitellata Hirudo medicinalis HCN Not cloned ? Regulates bursting activity of heart interneurons (Angstadt and Calabrese, 1989) *Only 8 of 18 cloned mammalia n HCN channels are shown.

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28 CHAPTER 2 CLONING AND MOLECULAR CHARACTERI ZATION OF THE acHCN CHANNEL 2.1 Introduction To study the properties and role of the acHCN channel it has first to be cloned from the CNS of A. californica A. californica belongs to a clade of animals called Lophotrochozoa (Peterson and Eernisse, 2001). Because no HCN channels have been cloned from any species of this group, a question arises about how acHCN differs from HCN channels of other lineages, specifically those belonging to two other clades: Ecdysozoa and De uterostomia (Gisselmann et al., 2003, 2005a, b; Ludwig et al., 1998). Thus, the major objective of the work presented in this chapter was to clone an HCN channel from the CNS of A. californica characterize the molecu lar organization of the channel, and compare it to the HCN channels from other organisms. Since only one HCN gene was found in the CNS of most invertebrates, my hypothesis was that it also holds true for A. californica. And because HCN channels of Ecdysozoa and Deuterostomia are highly conserved, I predicted that the general organi zation of acHCN would be similar to HCN channels of other organisms. However, some characteristics of acHCN might be different as a result of parallel evolution of the lophotrochozoan lineage. By cloning the A. californica HCN channel I could to dete rmine the distribution of the transcript in the CNS of the animal, express th e channel in a heterologous system and study the biophysical and pharmacological properties of acHC N in an environment without an interference from other channels, as well as in its native neuronal environment. Obtaining the sequence of acHCN also allowed me to perform comparativ e analysis of HCN channels across all three clades of the animal kingdom.

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29 2.2 Materials and Methods 2.2.1 Materials and Reagents All materials and reagents used for the work described in this and subsequent chapters were obtained from Sigma-Aldrich (St. Louis, MO) unless noted otherwise. 2.2.2 Cloning and Sequence Analysis Amplified cDNA was prepared from the w hole CNS, buccal, or pleural ganglia, as described previously (Matz, 2002). Primers (5-CGGAGTCTACGTCAGAGGATACC-3; 5-GACGATGCTGGGGTTCTTGCCGA3) were designed to the HCN-like cDNA sequence from our A. californica expressed sequence tag (EST) da tabase (Moroz et al., 2006). The obtained fragment was extended by multiple 5and 3-RACE (rapid amplification of cDNA ends) reactions (Matz et al., 1999). The complete coding region of acHCN was obtained from the CNS library by a polymerase chain reacti on (PCR) using the following primers: 5'-ATGGGGCAGGAATGCG TGGCTGGA-3' and 5'-TTAAGGATCTGGTCCTTGAGATAG CCGGT-3'. The products of each reaction were run on 1% agarose gel. Distinct bands were exci sed. The cDNA was purified using a Gel Extraction kit (Qiagen, Valencia, CA), ligated into pCR4 -TOPO vector (Invitrogen, Carlsbad, CA), and transformed into One-Shot competent E. coli cells (Invitrogen). The clones were isolated, purified using a MiniPrep kit (Q iagen) and sequenced by the Whitney Laboratory molecular core facility or by SeqWright (Houston, TX). Most of the HCN sequences used for alignments and construction of the phylogenetic trees were obtained from the Entrez Protein database (http://www.ncbi.nlm.nih.gov/sites/ entrez?db=Protein). Sequences of HCN transcripts for Lottia gigantea (Mollusca, Gastropoda), Nematostella vectensis (Cnidaria, Anthozoa), Capitella sp

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30 (Annelida, Polychaeta), and Ciona intestinalis (Urochordata, Ascideacea) were downloaded from the Joint Genome Institute (JGI) website (htt p://www.jgi.doe.gov/) following a search using the keyword hyperpolarization and translated using th e ExPASy Translate tool (http://www.expasy.ch/tools/dna.html). Each of the output sequences was then blasted against the NCBI database (http://www .ncbi.nlm.nih.gov/BLAST/) to ensu re their identity as HCN channels. The sequences confirmed to correspond to HCN channels were th en blasted against the JGI database for a particular organism to fi nd similar fragments and potentially extend the existing sequences. Alignment of HCN proteins from differe nt organisms was done using ClustalX (ftp://ftp-igbmc.u-strasbg.fr/pub/Cl ustalX/) with default paramete rs and the graphical output was produced by GeneDoc (http://www.nrbs c.org/gfx/genedoc/gddl.htm). The pore region, CNBD and transmembrane domains were predicted using SMART (http://smart.embl-heidelbe rg.de/index2.cgi) and TMPred (www.ch.embnet.org/software/T MPRED_form.html) respectively, and putative phosphorylation and glycosylation sites were obtained using PR OSITE (www.expasy.org/cg i-bin/scanprosite). The bootstrapped neighbor-joining phylogenetic tree was crea ted in ClustalX after removing all gaps in the alignment of HCN proteins (see Appendix A) or CNBD-containing potassium and cation channels (s ee Appendix B) in GeneDoc. Un rooted trees were generated because creating a rooted tree re quires knowing the ances tor of the group of analyzed proteins, and this ancestor is not know n for HCN channels. Boostrapping was done to determine the consistency of taxon bipartitions. Bootstrap values we re calculated as the number of times that a particular node appeared duri ng 1000 iterations while constructi ng the phylogenenetic tree. The graphical output was ge nerated using TreeView

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31 (http://taxonomy.zoology.gla.ac .uk/rod/treeview.html). Two HCN channels of N. vectensis were not put on the tree because they are too diverg ent and do not align with other HCN channels. 2.2.3 Determination of the Genomic Organization of acHCN To determine the genomic organizat ion of acHCN, the consensu s sequence of the acHCN clone (cDNA) was blasted against the trace archive of A. californica genomic sequences (~ 5x coverage). Regions of considerable sim ilarity (alignment scores 80 and higher) between the genomic sequences and the acHCN cDNA were considered to be exons and the genomic sequences flanking exons were considered to be introns. 2.2.4 Northern Blot 2.2.4.1 RNA isolation and blotting Total RNA was isolated from the whole A. californica CNS using the RNAaqueous (Ambion, Carlsbad, CA, USA) isolat ion protocol. Briefly, the cells were first disrupted in a lysis solution containing guanidinium thiocyanate; RNA was then bound by a silica-based filter and eluted in a small volume of elution solution. mRNA was obtained from the total RNA using a Poly(A) Purist kit (Ambion) based on binding of mRNA by Oligo(dT) Cellulose. RNA quality control was performed using a Bioanalyzer 2100 (A gilent, Santa Clara, CA). RNA samples were denatured by incubation for 30 min at 50C in Glyoxal Load Dye (Ambion) and then run on a gel made with Ambions Gel Running Buffer for 1.5 hrs at 5 V/cm. To transfer RNA from the gel to a membrane, transfer material was arrange d as follows (from the bottom): 3 cm of paper towels, three dry pieces of filter paper (Whatm an International Ltd., Maidstone, England) and two more pieces pre-wet in Transfer buffer (Ambion), nylon membrane (Micron Separations Inc., Westborough, MA) pre-wet in Transfer buffer, gel, and three more pieces of filter paper. RNA was transferred by cen trifuging the transfer material for 50 min at 500 rpm. The efficiency of transfer was checked by examining the membrane and gel under UV illumination. RNA was

PAGE 32

32 then cross-linked in a UV crosslinker 1800 (Str atagene, La Jolla, CA) and the cross-linked membrane was kept at -20C. 2.2.4.2 Chemiluminescent detection The membrane was pre-hybridized for 1 hr at 68C in 10 ml of ULTRAhyb buffer (Ambion). Antisense RNA probe (1 g/ l) was synthesized and labele d with DIG as described in section 4.2.3. 1 l of the probe was mixed with 1 ml of ULTRAhyb buffer and transferred to prehybridization solution. Hybridiz ation proceeded for 14 hrs at 68C. After hybridization, the membrane was washed once for 10 min with Low Stringency Wash Solution #1 (2x SSC, 0.1% SDS) (Ambion) using agitation and twice for 15 min with High Stringency Wash Solution #2 (0.1x SSC, 0.1% SDS) (Ambion) using agitation. Th e membrane was washed briefly (1-5 min) in Washing Buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% (v/v) Tween 20, pH 7.5) and incubated for 30 min in Blocking Solution (1x Bloc king Reagent) (Roche) in Maleic Acid Buffer 0.1 M maleic acid, 0.15 M NaCl, pH 7.5) and Antibody Solution (Alkaline-Phosphataseconjugated Anti-DIG antibodies di luted 1:20,000 in Blocking Solution). The membrane was then washed for 5 min in Washing Buffer and equilibrat ed for 5 min in Detection Buffer (0.1 M TrisHCl, pH 9.5, 0.1 M NaCl). The membrane was placed RNA side facing up on a development folder and CDP-Star (chemiluminescent substr ate for alkaline phosphatase) (Roche) working solution (5 l CDP-Star in 0.5 ml Detection Bu ffer) was applied. The membrane was immediately covered with another sheet of develo pment folder to spread the substrate evenly over the membrane, exposed to Hyperfilm EC L (high performance ch emiluminescence film) (Amersham Biosciences, Piscataway, NJ) fo r 1-5 min under safe-light illumination and developed in Konika SRX-101 developer. To strip the membrane of a probe it was inc ubated in Stripping Solution (50% formamide, 5% SDS, 50 mM Tris-HCl, pH 7.5) two times for 60 min at 80C, then rinsed in 2x SSC for

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33 5 min at room temperature and st ored in 2x SSC before re-using. 2.2.4.3 Radioactive detection The transcription/labeling reaction was performed as described in the Strip-EZTM RNA protocol (Ambion) in the presence of 60 Ci of labeled uridine ([ -32P]UTP) for the 533 nucleotide (nt) acHCN probe or 100 Ci for the whole (approximately 2 kb) acHCN probe and 1755 nt FMRFamide probe (corresponding to the whole FMRFamide gene) in 20 l total volume. Hybridization was performed in 10 ml of UltraHyb buffer. In each case, 10 l of probe was added giving a final radioactivity of 30 Ci for the 533 nt acHCN probe and 50 Ci for the whole acHCN and FMRFamide probes. H ybridization proceeded for 15 hrs. Development was carried out for 1-3 days in a cassette with enhancing screen at -80C using BioMax XARfilm (Kodak, Rochester, NY). 2.3 Results 2.3.1 Identity and Molecular Organization of acHCN One of my major goals was to clone the acHCN channel. It proved to be a difficult task due to both the low abundance of the HCN transcripts in A. californica neurons and the unusual organization of the 5and 3-regions of acHCN. By aligning and analyzing 168 cloned cDNA se quences, each one co rresponding to an HCN channel, I determined that I have cloned a coding region of the channel from the CNS of A. californica (Figure 2-1). The cloned channel is very similar to HCN channels from other organisms, which indicates that it indeed belo ngs to the HCN channel family (Figure 2-2). Following identification of a transcript, two ma jor questions arise: how many isoforms of the transcript are present in the organism, and was the whole reading frame obtained ?

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34 I found only one HCN channel in A. californica Existence of a si ngle HCN channel is supported by the fact that all 13 existing HCN ESTs from the A. californica transcriptome (Moroz et al., 2006) perfectly align with the co nsensus sequence of the cloned channel. Also, fragments corresponding to a sing le HCN channel have been f ound in the sequenced genome of A. californica (~5x coverage). In addition, several lines of evidence suggest that I obtained a clone containing the entire open reading frame (ORF). First, there is a stop codon (TAG) upstream from the first ATG supported by all nine sequences for this region. Second, there is a stop codon (TAA) at the end of the acHCN transcript. It is also supported by all sequences (n = 16) for this region. Third, the ORF of acHCN ends where HCN ch annels from most other organi sms end (Figure 2-2). Finally, the cloned channel has all the domains of other HCN channels: six TM, a pore region with a modified potassium selectivity filter, a voltage sensor in TM4 and a CNBD. Although acHCN is very similar to the HCN chan nels from other organisms, especially in its middle portion, the 5-region of the cloned ch annel is different from other HCN channels. acHCN does not start where most other HCN channe ls start (first black box in the alignment shown in Figure 2-2). It has an isoleucine at this position wh ereas HCN channels of other organisms have their first methionine (start). The first methionine of acHCN is 28 amino acids (aa) downstream. acHCN also has an in-frame alternative start codon CTG (Tee and Jaffe, 2001; Touriol et al., 2003) 65 aa upstream from its first methionine. Similar to other HCN channels, acHCN has eight positively-charged residues, seven arginines and a lysine in its vol tage sensor (Figure 2-3). Additi onally, as in other HCN channels, the potassium selectivity motif (TXXTXGYG) of acHCN is modified. While the GYG sequence is preserved, the first threonine residue is chan ged to histidine and the second threonine residue

PAGE 35

35 is changed to cysteine. The cons erved aspartate, which follows the GYG motif in most of the classic voltage-gated K+ channels, is altered in acHC N to arginine (Figure 2-4). Some studies suggest that HCN channel activity can be regulated by phosphorylation and dephosphorylation by a number of protein kinases (Vargas a nd Lucero, 2002; Wu and Cohen, 1997). I have predicted 14 putativ e phosphorylation sites for seve ral protein kinases including seven sites for casein kinase II ([ST]-x(2)-[DE] ), six sites for protein kina se C ([ST]x[RK]), and one site for tyrosine kinase ([RK]-x(6)-Y). One potential N-glycosyl ation site (N-x-[ST]) and seven N-myristoylation sites (G-{EDRKHP FYW}-x(2)-[STAGCN]-{P}) were also found (see Figure 2-2). However, because proteins ar e myristoylated only at their N-termini (MaurerStroh et al., 2002), the nu mber of predicted N-myristoylation sites in acHCN can be limited to three, which are at the N-terminal region of the protein at the positions 76, 77 and 110. The presence of 14 putative phosphorylation sites ma y provide ways, in addition to regulation by cAMP/cGMP, to link the activity of the channel to different signaling pathwa ys inside the cells. Having the sequence of acHCN, I constructed a phylogenetic tree of HCN channels from different vertebrate and invertebra te species (Figure 2-5). It show s that acHCN is closely related to other invertebrate HCN channels, part icularly those of a Prosobranch mollusk, Lottia gigantea (giant owl limpet), and a polychaete, Capitella sp High bootstrap values support grouping of acHCN with HCN channels of other Lophotroch ozoa. Together with molecular data, the phylogenetic tree provides additional evidence th at the cloned channel is in fact an HCN channel. Unlike vertebrates, which have four HCN ch annels, HCN1-HCN4, most invertebrates have a single HCN channel that may have several sp lice forms (Gisselmann et al., 2005a; Ouyang et al., 2007). A sea urchin, S. purpuratus has two HCN channels that were cloned from its sperm cells (Gauss et al., 1998; Galindo et al., 2005). I also found two HCN genes in the genome of the

PAGE 36

36 cnidarian N. vectensis and three HCN genes in the ascidian C. intenstinalis Existence of multiple HCN genes in animals of different phyla suggests that duplication of HCN genes occurred in these animals. 2.3.2 Duplication of a Coding Re gion at the 3-UTR of acHCN acHCN has a long 3-UTR which is almost identic al to the coding region of the transcript, but is frame-shifted and thus is not expected to be translated. The dupl icate sequence in the 3UTR was discovered when a fragment produced by one of the 5-RACEs aligned to the 3-end of the existing consensus seque nce of acHCN (Figure 2-6A). To confirm the existence of the duplication I de signed a direct primer at the 3-end of the transcript and a reverse primer at the 5-end of the transcript corresponding to the 5-end of the duplicate sequence. PCR with these primers produced a fragment that consisted of the 3-end of the HCN transcript and the 5-end of the duplicate sequence (Figure 2-6B). The same sequence was obtained from three di fferent libraries made from the whole A. californica CNS, buccal ganglia or B2 neuron in buccal ganglia. However, despite considerable effort (I performed 173 3-RACEs), the duplicate sequence was not read to the end and a poly-A sequence was not reached. There are two possible explanations for this. First, a long sequence containing both the coding region of acHCN and the 3-UTR ending with a polyA tail, may be very rare or non-existent in the available A. californica cDNA libraries. Second, during the production of the cDNA lib raries the acHCN fragments mi ght have terminated at the A-rich region at the 3-end of the transc ript (AAACAGAAGAAAAAGA CA), downstream of the duplicate sequence, because the poly(T)-contai ning adaptor, used to make these libraries, bound to this region. The latter explanation is supported by the fact that the 3-end of the transcript downstream to the A-rich region c ould not be obtained usi ng conventional 3-RACEs. It was read only in the expe riment shown in Figure 2-6B.

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37 Completion of the A. californica genome will help to determine the end of the acHCN transcript. However, having the whole coding region of acHCN I could then proceed to characterize the channel despite not obtaining the full 3-UTR. 2.3.3 Genomic Organization and Possi ble Splicing Variants of acHCN Determining the genomic organization of the channel provides addi tional means, besides analyzing expressed sequences, for the comparativ e and evolutionary study of HCN channels. By aligning the consensus cDNA sequence of acHCN and genomic sequences of A. californica I determined that the acHCN gene consists of at least 12 exons (Figure 2-7). The exact genomic organization of acHCN cannot be established at this time because, at the current stage of A. californica genome sequencing (~ 5x coverage), there ar e still gaps in genomic representation of the acHCN transcript, specifically in the 5 (nucleotides 211-439) and middle (nucleotides 1084-1301) portions of the transcript. The number of exons in the acHCN gene is sim ilar to that of arthr opods that have 13 exons. HCN genes of chordates have fe wer exons than invertebrate ge nes (Figure 2-8). Because the boundaries of most exons in HCN channels of vertebrates and invertebrates coincide, this suggests that exons in Chordata have fused. Current exon-intron junctions of acHCN are s hown in Table 2-1. There are at least seven potential variants of one of the exons, exon 9 (Table 2-2), comprisi ng the beginning of the CNBD. These variants differ in nucleotides in f our positions (Figure 29). Two variants were found among both genomic and cDNA sequences (var iants 1 and 2 in Table 2-2). They differ by the presence or absence of an ad enine in one of the positions. The presence of a nucleotide at this position leads to a frame shift and an app earance of a stop codon (TGA) 36 nucleotides downstream. There is a contig (a set of overlapping DNA segments derived from a single genetic source) in the preliminary A. californica genomic assembly that contains both of these variants

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38 next to each other. It su pports the presence of both of these variants in the A. californica genome and suggests that they may be used by alternative splicing. Interestingly, two splice variants also exis t at the beginning of the CNBD in the HCN channel of the spiny lobster, Panulirus interruptus (Ouyang et al., 2007), at approximately the same position as in the A. californica HCN channel (they start six amino acids downstream compared to acHCN and end two amino acids downstream). These two variants differ by amino acids in seven positions. In the 5-most of these positions the HCN channel of the spiny lobster has either tyrosine or phenylalanine, while acH CN has either a positively-charged histidine or negatively charged glutamine. This is the onl y difference between the putative variants of acHCN that is expected to change the properties (charge) of this region of the channel, because three other nucleotide changes are conservati ve. Also, this is th e only position containing alternative amino acids in both A. californica and P. interruptus suggesting that it may be important in determining the differential properties of these splicing variants. Alternative splicing in the same region of both A. californica and P. interruptus HCN channels suggests that different splice variants at this region may be produced to regulate modulation of the channel by cyc lic nucleotides. In support of th is hypothesis, Ouyang et al. (2007) showed that the two alternative variants of the spiny lobster are regulated differently by cAMP. 2.3.4 Northern Blot Because acHCN starts at a different position than HCN channels of other organisms I decided to verify the length of acHCN and determ ine if forms of the transcript other than the consensus one are synthesized. A traditional method to resolve these issues is Northern blot. I performed 18 experiments using two different probes to acHCN. Probes to a serotonin

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39 transporter (gi:15282076) or FMRFamide (gi:155762) were used as controls since these are among the most abundant transcripts in the A. californica CNS. I performed two types of experiments usi ng either chemiluminescent labeling (with a serotonin transporter probe serving as a control) or radio active labeling using -32P-UTP (with an FMRFamide probe serving as a control). Using a st andard Northern blot protocol (see section 2.2.4) I did not detect any acHCN bands. To increase the HCN signal I used the following variations of the protocol: 1 Placed on the membrane either the total RNA isolated from the whole A. californica CNS or mRNA, obtained from the total RNA. The latter was done to increase the sensitivity of the assay because mRNA makes up only 2 % of the total RNA. 2 Used either a 533 nt probe corresponding to the middle portion of the transcript or a 2 kb probe corresponding to the whole transcript to determine which probe will give a better signal. The shorter probe is expe cted to bind to its target be tter, while the longer probe is more specific. 3 Increased probe quantity up to 10 times (for chemiluminescent detection). 4 Increased antibody concentration up to 1:10,000 (for chemiluminescent detection). 5 Extended incubation time in antibodies up to 1 hr (for chemiluminescent detection). 6 Extended hybridization time to either 24 or 41 hrs. 7 Decreased hybridization temperature from +62C to + 58C to lower the stringency of hybridization and incr ease sensitivity. 8 Increased development time up to 3 weeks (for radioactive detection). 9 Carried out development in a phosphoimager that is more sensitive than film (for radioactive detection). Each time a control band (of the serotonin transporter or FMRFamide) of the expected length was clearly visible, but no HCN bands were detected (Figure 2-10). Possible reasons for this will be discu ssed in section 2.4.8.

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40 2.4 Discussion 2.4.1 Identity and Molecular Organization of acHCN acHCN is the first HCN channel cloned from the Lophotrochozoa clade. The presence of HCN channels in all three cl ades of bilaterian animals: Ecdysozoa, Lophotrochozoa and Deuterostomia, as well as in Cnidaria, suggests th at it was present in their common ancestor, and that it was possibly lost in nematodes. acHCN is very similar to HCN channels from other organisms with the most similarity to the HCN channels cloned from A. mellifera (86 % identity), Heliothis virescens (tobacco budworm) (84 %), Aedes aegypti (mosquito) (84 %) and P. argus (83 %). Of human HCN channels, acHCN is most similar to the HCN3 (73 % identity). The similarity of the voltage sensor, pore region and CNBD of acHCN and ot her HCN channels suggests that acHCN has similar voltage dependence, permeability and regulation by cyclic nucleotides. The major difference of acHCN is in the leng th of its 5-region. In the position where HCN channels of most other animals have their firs t ATG (start codon) acHCN has an ATT sequence. This difference is most likely due to a singl e nucleotide mutation (G to T supported by all 12 sequences for this region) resulting in a loss of the first methionine and possible truncation of the channel. However, the region of acHCN between the ATT and the first ATG codon is similar to the corresponding region of other HCN cha nnels. In addition, acHCN has two predicted phosphorylation sites in this region. Both of these facts suggest that this region may be important in the channels functioning. Conservation of the sequence between the ATT and the first ATG of acHCN suggests that this regi on is translated because untransl ated regions are modified quite rapidly. Translation of acHCN ma y start from an upstream alternative start codon CTG. In this case all similarity of the 5 -end of acHCN and other HCN channels would be preserved. Otherwise, if translation of the channel starts from the current first ATG codon, the

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41 aforementioned facts can be explaine d by the recent occurrence of the ATG ATT mutation. In this case, there was not enough evolutionary tim e for the untranslated region between the ATT and first ATG sequences to undergo significant cha nges. To determine the translation start of acHCN, Western blot using anti bodies to the acHCN protein ha s to be performed, but these antibodies currently do not exist. The similarity of acHCN and HCN channels fr om other organisms implies that they may have similar biophysical and pharmacological prope rties, but differences in the 5-region may result in some unique characteristics of acH CN discussed in more details in Chapter 3. The similarity of the CNBD of the analyzed HCN channels and othe r cyclic nu cleotideregulated proteins and the exis tence of many common structural elements with voltage-gated potassium channels suggests that HCN channels originated from a potassium channel that acquired a CNBD. To test this hypothesis and determine how HCN chan nels changed in the course of evolution I will examine HCN channe ls of more basic organisms, starting with invertebrate animals. 2.4.2 HCN Channels in Invertebrates Of 34 phyla containing invertebrate animal s, HCN channels have been cloned and characterized in species belonging to only two phyla: Echinodermata ( Strongylocentrotus purpuratus ) and Arthropoda: Drosophila melanogaster Apis mellifera Panulirus argus and P. interruptus (see Table 1-1). By analyzing recently emerged genomic and transcriptomic data of different invertebrate animal s I found HCN channels in species of four other phyla: Cnidaria ( Nematostella vectensis ), Annelida ( Capitella sp. ), Mollusca ( Lottia gigantea ) and basal Chordata (Urochordata) ( Ciona intenstinalis ). Of the animals with completed genomes only nematodes Caenorhabditis elegans and C. briggsae lack HCN channels, although they have related cyclic nucleotide-gated (CNG) channels (Figure 2-11).

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42 The presence of two HCN genes in Cnidaria suggests that an ancestral bilaterian organism also had two HCN genes; there was a gene loss before the divergence of Ecdysozoa and Lophotrochozoa, a duplication before the diverg ence of Urochordata and other Chordata and another one in vertebrates (Fi gure 2-12B). By a different scen ario, an ancest ral bilaterian organism had a single HCN gene. Duplications o ccured independently in Cnidaria, before the divergence of Deuterostomes and Ecdysozoa / L ophotrochozoa, Urochordata and other chordates and another one in vertebrates. This second possi bility suggests that a gene loss occurred only once, in nematodes (Figure 2-12A). Although nothing is known about HCN channels in the other 28 phyla of the animal kingdom, the molecular organization and/or pr operties of known HCN channels in seven different phyla are very similar. This suggests that HCN channels emerged very early in the evolution of animals or even before the divergen ce of animals. To determine which scenario is true, I next screened for HCN or rela ted channels in non-animal eukaryotes. 2.4.3 Cyclic Nucleotide-Binding Cha nnels in Non-Animal Eukaryotes I did not find HCN channels in any non-anim al species. However, plants have a similar family of channels, called cyclic nucleotidegated cation channels (C NGCs, see Figure 2-11). These channels were isolated from thale cress ( Arabidopsis thaliana) (AtCNGC, Kohler, 1999), barley ( Hordeum vulgare ) (HvCBT1, Schuurink et al., 1998), rice ( Oryza sativa ) and tobacco ( Nicotiana tabacum ) (NtCBP4, Arazi et al., 1999). Like animal CNG channels, plant CNGCs are permeable to Ca2+, blocked by external Ca2+, and have a calmodulin-binding domain. But while animal CNG channels conduct mostly Ca2+ and Na+ under physiological conditions, plant CNGCs are strongly selective for K+ over Na+ (Leng et al., 2002). Whereas animal CNG channels are activated by cGMP and only weakly by cAMP, the latter strongly activates plant CNGC channels. In addition, hyperp olarization is required for th eir activation. These properties

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43 relate plant CNGCs to animal HCN channels. However, plant CNGC channels lack the GYG potassium selectivity filter. Instead, specific amino acids w ithin their pore (Asn416 and Asp417 in AtCNGC2) facilitate K+ over Na+ conductance (Hua et al., 2003). This may indicate independent origin of plant CNGC channels and animal CNG / HCN channels or different demands on plant cells. Plants also have potassium-selective, vo ltage-gated channels containing a cyclic nucleotide-binding domain, including KAT1 (And erson et al., 1992), AKT 1 (Sentenac et al., 1992) and KST1 (Muller-Rober, 1995) (see Fi gure 2-11), but contra ry to HCN and CNG channels, binding of cyclic nucleot ides decreases their current, making them similar to animal ether-a-go-go-related (ERG) channels (Cui et al., 2000) (Table 2-3). Protozoa ( Paramecium aurelia ) have a similar cyclic nucleotide-binding pot assium channel, while Fungi lack channels with a CNBD (I checked 15 species with completed genomes, including Aspergillus fumigatus Candida glabrata Cryptococcus eoformans Debaryomyces hansenii Encephalitozoon cuniculi Eremothecium gossypii Gibberella zeae Kluyveromyces lactis Magnaporthe grisea Neurospora crassa Pichia stipitis Saccharomyces cerevisiae Schizosaccharomyces pombe Ustilago maydis Yarrowia lipolytica ). Absence of HCN channels in Fung i, a sister group of animals, suggests that these channels first evolved in animals. However, to determine what channels gave rise to HCN and animal and plant cyclic nucleotide-gated pota ssium channels, I screened for potassium channe ls containing a CNBD in prokaryotes. 2.4.4 Cyclic Nucleotide-Binding Po tassium Channels in Prokaryota I found five putative cyclic nuc leotide-binding potassium cha nnels in Prokaryotes (see Figure 2-11). All of these channels have the classic K+ selectivity motif, which suggests that they conduct predominantly potassium ions.

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44 Archaea do not have cyclic nucleotide-binding channels, but a potassium channel activated by hyperpolarization has been characterized in Methanococcus jannaschii (Sesti et al., 2003). The putative cyclic nucleotide-binding potassium channel of Rhodopseudomonas palustris has five regularly interspaced positively-charged residues in its TM4 domain, Trichodesmium erythraeum has four, Magnetospirillum magnetotacticum has three and Bradyrhizobium japonicum and Mesorhizobium loti have two. This suggests that th ese channels are also regulated by voltage, though to a lesser degree than HCN cha nnels. Also, the CNBD of these channels is very similar to the CNBD of bacterial catabol ite activator protein (C AP). Thus, prokaryotic cyclic nucleotide-binding potassium channels mi ght have appeared when a potassium channel acquired a CNBD of another protein. Analysis of HCN and related channels allo ws making a hypothesis about the origin and potential evolution of these channels. 2.4.5 Origin and Potential Evolution of HCN and Related Channels Comparative analysis of transcripts containi ng a cyclic nucleotide-binding domain shows that the CNBD of HCN and rela ted channels from plants, Prot ozoa and Prokaryota are very similar to the CNBD of bacter ial CAP and PKA (Finn et al., 1 996; Zagotta et al., 2003). This suggests that these channels evolved by a pot assium channel acquiring a CNBD of another protein. The similarity in the molecular organizat ion of HCN channels and CNBD-containing potassium channels of Prokaryota, including th e presence of six TM, the GYG sequence, and a conserved CNBD suggests that HCN channels evol ved from cyclic nucleotide-binding potassium channels of ancestral prokaryotic organisms by pr eserving mutations in the potassium selectivity filter, specifically threonines preceding GYG and aspartate following the GYG sequence. These modifications allow Na+ to pass through the ch annel pore along with K+, setting the reversal

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45 potential of the channels at more depolarized values and thus determining pacemaker properties of spontaneously active cells. P acemaker cells play a crucial ro le in coordination of neuronal activity in central pattern gene rators, which are responsible fo r rhythmic behaviors such as heartbeat in leech (Cymbalyuk et al., 2002), or feeding in Lymnaea (Straub and Benjamin, 2001), as well as in intercellular si gnaling and synchronization in the brain (Maccaferri and McBain, 1996; Bal and McCormick, 1997; Luthi et al., 1998). Thus, HCN channels might have evolved to control integrative f unctions of animals. However, poor bootstrap support of branching of different classes of cation and potassium cyclic nucleotide-binding channels and lack of electrophysiological data from prokaryotes prevents making definite conclusions about the HCN channel origin and evolution. For example, it is not known whether prokar yotic CNBD-containing potassiu m channels are activated by depolarization or hyperpolarizat ion and whether they are activ ated or inhibited by cyclic nucleotides like HCN channels or plant vol tage-gated potassium channels, respectively. Expression and functional character ization of prokaryotic cyclic nucleotide-bind ing potassium channels as well as HCN channels from basi c eukaryotic organisms will provide a more complete picture of these issues. 2.4.6 Duplication of a Coding Region at the 3-UTR of acHCN and Genomic Organization of the Channel The similarity of the 3-UTR and the c oding region of acHCN suggests that the A. californica HCN gene has undergone duplication (or mu ltiple duplications). This duplication must have been recent because of the near identity of the coding region and duplicate sequence and the fact that the latter did not acquire any independent regulat ory elements and is transcribed together with the coding region. Multiple duplica tion events might have also resulted in the presence of different variants of exon 9 of acHCN.

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46 2.4.7 Possible Splice Variants of acHCN The existence of seven variants of exon 9 s uggests that up to seven splice forms of acHCN channel are synthesized that may differ in thei r function. The variant with a frame shift and a putative stop codon may produce a shortened fo rm of the acHCN chan nel without a CNBD (Figure 2-13). It is recognized that the CNBD has an inhibitory effect on the channel activity and binding of cyclic nucleotides reli eves this inhibition. Deletion of the CNBD makes the channel constitutively-active (Wainger et al., 2001), i.e., voltage dependence of Ih activation strongly shifts to a depolarizing direction as in the pr esence of saturating c oncentration of cyclic nucleotides. The putative shortened form of th e acHCN may be a natura l analog of the CNBD deletion. This form is expected to be constantly activated a nd thus may participate in the development of LTP and LTH. Determining the cond itions at which this form is synthesized is part of the future work. 2.4.8 Verification of Channel Length To determine the exact length of acHCN and how many copies of the coding region sequence are present in the 3-U TR I performed Northern blot. This method did not detect any HCN bands. The presence of the control bands (s erotonin transporter or FMRFamide) eliminates the possibility of a mistake in the design and exec ution of the experiments since both control and acHCN experiments were performed simultaneously using the same protocol and reagents. Also, I verified that the acHCN probe was functional by its successful use in in situ hybridization experiments. Thus, the reason that I did not detect any HCN bands is almost certainly due to the low abundance of the acHCN transcripts that is below the sensitivity limit for Northern blot. In situ hybridization provides independent evid ence supporting this conclusion. Abundant transcripts (e.g., FMRFamide, serotonin tran sporter) develop in 5-10 minutes while HCN channel transcripts take approxi mately 2 hrs. Because development time is considered to be

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47 proportional to the abundance of a transcri pt, the acHCN RNA is indeed rare in A. californica neurons. In conclusion, verification of the length of acHCN is difficult because of a low abundance of the transcript. Completion of the A. californica genome sequencing will help to resolve this issue. 2.4.5 Summary The evidence presented in section 2.3.1 imp lies that the whole coding region of acHCN was cloned. This channel is very similar to HC N channels from other organisms. However, possible truncation of the 5-end of acHCN still leaves a question of whether the channel is functional. This question is a ddressed in Chapter 3 by expressi ng the channel in a heterologous system and studying its biophysical and pharmacol ogical properties. Obtaining a sequence of acHCN also allowed localization of the channel transcript that is described in Chapter 4. Because specific antibodies to acHCN have not been generated, this chapter does not deal with the acHCN protein. In the future, we plan to obtain these antibodies and localize the acHCN protein in the CNS of A. californica using immunocytochemistry. We also plan to investigate whether translation of acHCN starts from the fi rst ATG or from the a lternative start codon CTG and whether a shortened form of acHCN without CNBD is synthesized in the A. californica CNS (and if so, under what conditions). The latter issues will be answered by performing Western blots with antibodies to acHCN.

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48 Figure 2-1. The longest cDNA se quence and predicted amino aci d sequence of acHCN. The cDNA sequence is a consensus of a 168 clone assembly. The ATG sequence coding for the first methionine (shown in blue box) is supported by 29 chromatograms, the stop codon upstream of the first methionine is supported by nine chromatograms, and the stop codon at the 3-end of the transc ript is supported by16 chromatograms (stop codons are shown in red boxes). The amino aci d sequence is shown starting from the alternative start codon CTG and ending at the stop codon. Predicted transmembrane domains (TM1-TM6), a pore and a cyclic nucleotide-binding domain (CNBD) are underlined.

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49 Figure 2-2. Alignment of HCN pr oteins from different organisms. The alignment shows that HCN channels are highly conserved, esp ecially in their TMs, pore and CNBD. The presence of 14 predicted phosphorylation s ites suggests potential regulation of the channel by phosphorylation by protein kinase s. The first conventional (ATG) and the alternative (CTG) start codons of acHCN are shown in red and the first ATG codon of other HCN channels is shown in blue The red bar shows a region of a weak alignment of acHCN and other HCN cha nnels upstream from acHCNs first methionine. Abbreviations: ac A. californica hs Homo sapiens (human), pa Panulirus argus (lobster), am Apis mellifera (honey bee). Predicted sites of phosphorylation by: Protein kinase C; Casein kinase II; Tyrosine Kinase; N-myristoylation site; N-glycosylation site CTG ATT ATG ATG CTG ATT ATG ATG CTG ATT ATG ATG CTG ATT ATG ATG

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50 Figure 2-3. Alignment of the voltage sensors from the HCN channels of different organisms. The alignment shows that acHCN, as well as other HCN channels, has eight positively-charged residues (red boxes) which are regularly interspaced. Conserved residues are shown in blac k boxes. Abbreviations: ac A. californica hs Homo sapiens (human), pa Panulirus argus (lobster), am Apis mellifera (honey bee).

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51 Figure 2-4. Alignment of the pore regions from th e HCN channels of different organisms. The alignment shows that the core region (re d box) of the potassium selectivity motif (shown above the alignment) is preserved in acHCN, as well as other HCN channels, but the following residue (shown in blue, green or orange) and the threonines preceding the GYG sequence are changed. C onserved residues are shown in black boxes. Abbreviations: ac A. californica hs Homo sapiens (human), pa Panulirus argus (lobster), am Apis mellifera (honey bee).

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52 Figure 2-5. Phylogenetic tree of HCN channels. The phylogenetic tree was created in ClustalX after removing all gaps in alignment of HC N proteins with GeneDoc. The graphical output was generated using TreeView. Numb ers at the nodes represent bootstrap values. Accession numbers: human HCN1 gi:32698746, human HCN2 gi:30580783, human HCN3 gi:38327037, human HCN4 gi:4885407, zebrafish ( Danio rerio ) HCN1 gi:94732421, HCN4 gi:125842901, sea urchin ( Strongylocentrotus purpuratus ) HCN1 gi:3242324, sea urchin HCN2 gi:69146514, Drosophila melanogaster HCN (splice form A1B1C1) gi:24653608, lobster ( Panulirus argus ) HCN gi:33355925, honey bee ( Apis mellifera ) HCN gi:52631748. Sequences of HCN channels of the limpet ( Lottia gigantea ), polychaete worm Capitella sp ., and the sea squirt ( Ciona intestinalis ) were downloaded from the Joint Genome Institute website.

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53 Figure 2-6. Experiments showing the existence of a duplicate sequence at the 3-end of acHCN. A) 5-RACE produced a fragment that ali gned to the 3-end of the consensus acHCN sequence. B) A PCR with a direct primer at the 3-end of the transcript (primer 1) and a reverse primer at the 5-end of the tr anscript (primer 2, corresponding to the 5end of the duplicate sequence) produced a fragment consisting of the 3-end of acHCN and the 5-end of the duplicate seque nce. C) Schematic diagram of the chimeric organization of acHCN. stop codons, ATG start codon, CTG alternative start codon. Numbers i ndicate numbers of nucleotides. Coding region CTG ATG 97 195 1881 2885-UTR 3-UTRDuplicate Seq C Coding region CTG ATG 97 195 1881 2885-UTR 3-UTRDuplicate Seq Coding region CTG ATG 97 195 1881 2885-UTR 3-UTRDuplicate Seq C

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54 Figure 2-7. Genomic organiza tion of acHCN. A) Schematic diagram of the exon-intron organization of the acHCN gene. B) Alignment of the acHCN cDNA with genomic sequences from the Trace Archive data base in NCBI BLAST.The acHCN gene consists of at least 12 exons shown in red (alignment score => 200) and pink (alignment score 80-200) bars in the positio ns corresponding to the consensus cDNA sequence (shown by the blue bar). 1 2 3 4 5 6 7 8 9 10 11 12A B

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55 Figure 2-8. Number of exons in HCN genes in different animal phyla. + sign following a number indicates that a gene consists of more than the specified number of exons, but an exact number cannot be determined due to the lack of geno mic data. Molluskan HCN genes most probably have 12 exons. Th is prediction is based on the assumption that the remaining gaps in the genomic representation of the channel transcript contain only one exon because these gaps have approximately the same size as known exons. However, these gaps may contain more than one exon. HCN genes of chordates have fewer exons than invertebrate ones. This suggests fusion of exons in Chordata. Available genomic data does not allow predicting the number of exons in Cephalochordata ( Amphioxus ). Cnidaria Chordata Urochordata Echinodermata Mollusca Annelida Arthropoda 6+ 8+ 10 8 8 8 8 14 15 9 11 12? 9+ 13 Cnidaria Chordata Urochordata Echinodermata Mollusca Annelida Arthropoda 6+ 8+ 10 8 8 8 8 14 15 9 11 12? 9+ 13

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56 Figure 2-9. Sequence of exon 9. Nucleotides, wh ich differ between splice variants of acHCN are shown by numbers 1-4. ACACGAGGTTGTGAACCA1AACTGTCG2TCTCTGGTG GCATCGGT3CCCTTCTTTACCAACGCCGATCC4GCCT TTGTGTCGGAGGTAGTGAGCAAGCTCAAGTTTGAGGT

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57 Figure 2-10. Comparison of Northern blots us ing probes to acHCN and FMRFamide (control). Total RNA isolated from the whole A. californica CNS was transfered to a membrane. Probes were labeled by -P*-UTP. No bands were detected for the HCN channel whereas a single band with the e xpected length of approximately 2 kb was visible for FMRFamide. The acHCN band was expected to be of approximately the same length. HCN FMRF2 kb HCN FMRF2 kb

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58 Figure 2-11. Phylogenetic tree of potassium and ca tion channels containing a cyclic nucleotidebinding domain. The phylogenetic tree was cr eated in ClustalX after removing all gaps in alignment of HCN proteins in Ge neDoc. The graphical output was generated using TreeView. Accession numbers: human HCN1 gi:32698746, human HCN2 gi:30580783, human HCN3 gi:38327037, hu man HCN4 gi:4885407, sea urchin ( Strongylocentrotus purpuratus ) HCN1 gi:3242324, sea urchin HCN2 gi:69146514, Drosophila melanogaster HCN (splice form A1B1C1) gi:24653608, lobster ( Panulirus argus ) HCN gi:33355925, human CNGA1 gi:2506302, human CNGA2 gi:2493743, human CNGA3 gi:13959682, human CNGB1 gi:2493750, human CNGB3 gi:48474982, D. melanogaster CNG gi:908846, Caenorhabditis elegans

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59 Figure 2-11. Continued CNG-1 (tax-2) gi:1805259, C. elegans CNG-2 (tax-4) gi:22096336, sea urchin CNG gi:72005966, Limulus polyphemus CNG gi:18657056, Arabidopsis thaliana CNG1 gi:15238657, A. thaliana CNG2 gi:38502856, A. thaliana CNG3 gi:18407073, A. thaliana CNG4 gi:38503128, A. thaliana CNG6 gi:38502863, A. thaliana CNG7 gi:15219100, A. thaliana CNG10 gi:38503202, A. thaliana CNG12 gi:38503031, A. thaliana CNG15 gi:38503241, A. thaliana CNG17 gi:38503044, A. thaliana CNG18 gi:38503201, A. thaliana CNG19 gi:38503198, A. thaliana CNG20 gi:38503198, tobacco CNG gi:6969231, rice CNG gi:50943023, A. thaliana AKT1 (cyclic nucleotide binding / in ward rectifier potassium channel) gi:15225768, A. thaliana AKT2 gi:18415864, rice AKT1-like gi:17887457, tobacco NKT2 gi:56744187, corn ZMK2 gi:5830781, carrot K+ channel gi:6562375, aspen ( Populus trichocarpa ) K+ channel gi:9955728, A. thaliana SKOR (cyclic nucleotide binding / outward rectifier potassi um channel) gi:15232991, human HERG gi:4156239, rat EAG7 gi:18777774, D. melanogaster ERG gi:16197863, C. elegans ERG gi:7739757, Paramecium aurelia K+ channel (PAK11) gi:17030694, Rhodopseudomonas palustris K+ channel gi:39651151, Bradyrhizobium japonicum K+ channel gi:27354026, Magnetospirillum magnetotacticum K+ channel gi:46202428, Mesorhizobium loti K+ channel gi:14023572, Trichodesmium erythraeum K+ channel gi:71674753

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60 Figure 2-12. Phylogenetic trees showing the nu mber of HCN genes in different phyla of the animal kingdom. These trees are based on th e assumption that an ancestral organism had either one (A) or two (B) HCN genes. A) Duplications (shown by +) occured in Cnidaria, before the divergence of Deuter ostomes and Ecdysozoa / Lophotrochozoa, Urochordata and other Chordata and in vertebrates. Gene loss (shown by x) occurred in nematodes. B) Duplications occurred before the divergence of Urochordata and other Chordata and in vertebrates. Gene loss occurred before the divergence of Ecdysozoa and Lophotrochozoa and in Nematoda. + + + +XCnidaria Chordata Urochordata Echinodermata Mollusca Annelida Nematoda Arthropoda + +XCnidaria Chordata Urochordata Echinodermata Mollusca Annelida Nematoda Arthropoda X A B + + + +XCnidaria Chordata Urochordata Echinodermata Mollusca Annelida Nematoda Arthropoda + +XCnidaria Chordata Urochordata Echinodermata Mollusca Annelida Nematoda Arthropoda X A B

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61 Figure 2-13. Two forms of the acHCN channe l that may be synthesized. If there is no nucleotide in position four of exon 9 (see Figure 2-9), then the whole channel, including the CNBD, will be tr anslated (A). The presence of adenine in position four of exon 9 produces a frame shift followed by a stop codon 36 nucleotides downstream. In this case a shortened form of the acHCN channe l without the CNBD may be synthesized (B). / A 5 HCN CNBD 3 */ A 5 HCN 3 No CNBD A B / A 5 HCN CNBD 3 */ A 5 HCN 3 No CNBD / A 5 HCN CNBD 3 */ A 5 HCN 3 No CNBD A B

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62 Table 2-1. Sequence and position of exon-intron junctions of acHCN gene Exon # Exon size, bp 3 intron junction Exon 5 sequence Exon 3 sequence Intron # 5-intron junction 1 211 GTG CCC CAC AAT TAT AAG 1 gt aata 2 228 ? GCC CAC GAC CTC ATC AAG 2 ? 3 61 ? ? ? CAA CTT CAG 3 ? 4 167 tatgcctcattttcttc ag GTT CTA CTG GGA CAG GCA 4 gt gcaa 5 165 attttttaaatctatgc ag TCA TCT TGA TCC ATG CAG 5 gt acgc 6 98 ttcctttttcggttgac ag GCC GAG CCT TGG GAA GAG 6 gt atgt 7 87 cttgcttgtactctcgc ag TTT CTA GCC GGG CTG CAG 7 gt acgt 8 282 ? TTC CTG GTG AAT GAA AAG 8 ? 9 147 ctctttcttctattttt ag TTC AAG CAA CTC AAA CAC 9 gt atgc 10 105 tgctgtattgactctac ag GAG GTT GTG AAG TTT GAG 10 gt aatt 11 140 caatgtacatctctctc ag GTG TAC CAG CTT TGG AGA 11 ? 12 441 cactaaccactcgac ag AAT CTG TCT GGA GAC GAA 12 ? *All shown intron sequences start with gt and e nd with ag. Sequences that can not be reliably discerned due to the lack of genomic da ta are substituted by question marks.

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63 Table 2-2. Identities of nucleotides in the positions 1-4 shown in Figure 2-9 Variant # Nucleotide 1 Nucleotide 2 Nucleotide 3 Nucleotide 4 Presence 1 C G A A cDNA, genome 2 C G A cDNA, genome 3 C G G cDNA 4 C C G cDNA 5 C C G G Genome 6 A G G Genome 7 A G A G Genome

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64 Table 2-3. Comparison of propert ies of HCN channels and othe r channels containing a CNBD Channel Class Voltage Activation cNMP Regulation Permeability HCN Hyperpolarization cAMP, cGMP Positive modulation K+, Na+ Animal CNG cGMP, cAMP Activation Na+, Ca2+ Plant CNG Hyperpolarization cAMP Activation K+, Na+, Ca2+ Plant K+ channels Depolarization cAMP Negative modulation K+ Either-a-go-go related Depolarization cAMP Negative modulation K+ Prokaryotic K+ channels ? cNMP ? K+ ?

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65 CHAPTER 3 BIOPHYSICAL AND PHARMACOLOGICA L CHARACTERIZATION OF acHCN 3.1 Introduction The molecular organization of acHCN de scribed in Chapter 2 left a question about whether the cloned channel is functional. Thus, two major objectives of the work presented in this chapter were to answer this question by determining th e properties of acHCN in an environment where other channels do not interfere, and to find an inhibitor of the channel for functional studies. Most characterized HCN channels have similar biophysical and pharmacological properties which reflect the similarity of th eir molecular organization (see Chapter 2). In response to hyperpolarization these channels generate slow inward currents that do not inactivate. The only exceptions are HCN channels in frog melanotrophs (M ei et al., 1998), and SpHCN1, one of the two HCN channels cloned from sperm flagella of the sea urchin S. purpuratus (Gauss et al., 1998). In addi tion to a slow steady-stat e current, SpHCN1 and the frog HCN channels have an instantaneous transient component. Because of modifications in their potassium selectivity motif, HCN channels also conduct Na+ (Heginbotham et al., 1994). Due to permeability to sodium ions the reversal potentials of HCN channels is more depolarized compared to classic K+ channels (Table 3-1). Alterations of the potassium selectivity motif may also acc ount for the slow kinetics of HCN channels compared to classic K+ channels. For example, in humans, the values of time constants of activation ( ) range from 196 ms to 23 s in HCN2 and HCN4 channels respectively (Table 3-1). Another common feature of all HCN channels is their activation by cyclic nucleotides, specifically cAMP and cGMP (Pape and Mc Cormick, 1989; Pedarzani and Storm, 1995; DiFrancesco and Tortora, 1991; Ludwig et al., 1998). They cause a depolarizing shift in the voltage activation of the channels, which may be as large as +41 mV (produced by 1 mM cAMP in P. argus ) (Gisselmann et al., 2005b), and, sometimes, in crease the current amplitude as in the

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66 mouse HCN1 channel (Santoro et al., 1998; Tabl e 3-1). The only known exception is the human HCN3 channel, which is not activated by cyclic nucleotides (Stieber et al., 2005). HCN channels are inhibited by low millimolar concentrations of Cs+ (Glitsch et al ., 1986) and a specific blocker ZD7288 (4-(N-ethyl-N-phenylamino)-1,2dimethyl-6-(methylamino) pyridinium chloride) (BoSmith et al., 1993) altho ugh in invertebrates the la tter was tested only in P. argus (Gisselmann et al., 2005b). Because acHCN has a predicted molecular stru cture very similar to the HCN channels cloned from other organisms, my hypothesis is that its majo r biophysical and pharmacological properties, specifically regulati on by hyperpolarization and cyclic nucleotides, slow activation and inhibition by Cs+ and ZD7288, will be similar. However, differences in the length of the Nterminal region of acHCN may conf er some unique properties to the A. californica channel. Characterizing the cloned acHCN in a heterologous system will confirm that it is a functional form of the channel and be a basis fo r identifying and characterizing the channel in A. californica neurons. 3.2 Methods 3.2.1 acHCN RNA Synthesis Two constructs were made from the coding re gion of a single acHCN transcript (Figure 3-1). Construct 1 started at th e alternative start co don (CTG) 201 nucleotides upstream from the first ATG codon of acHCN and ended at the stop codon at the 3-end of th e transcript. It was obtained from A. californica CNS library by a PCR using the following primers: 5'-CTGGGACCAACTAGTGGCGCCGGGA-3' and 5'-TTAAGGATCTGGTCCTTGAGATAGCCGGT-3'. Co nstruct 2 started at the first ATG codon of acHCN and ended at the st op codon at the 3-end of the transc ript. It was obtained from the same CNS library by a PCR using the following primers:

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67 5'-ATGGGGCAGGAATGCG TGGCTGGA-3' and 5'-TTAAGGATCTGGTCCTTGAGATAGCCGGT-3'. These constructs were cloned into LingT plasmid a modified BlueScript plasmid engineered to include 5and 3-UTRs of the Xenopus laevis -globin to facilitate expression in frog oocytes, presumably by increasing RNA stability (Jespersen et al., 2 002) (Figure 3-1). The constructs were transformed into One-Shot competent E. coli cells (Invitrogen). The clones were isolated, purified using a MiniPrep kit (Qiage n) and sequenced by the Whitney Laboratory molecular core facility. cRNA for oocyte injections was obtained by in vitro transcription of PmeI-linearized LingT plasmid using mMessage mMachine T7 Ultra (Ambion), a high yield, capped RNA transcription kit. The integrity and quantity of the acHCN cRNA were determined by a 2100 Bioanalyzer (Agilent). 3.2.2 Expression in the Xenopus Oocytes Mature (>9 cm) female African clawed frogs X. laevis (Xenopus Express, Plant City, FL) were used as a source of oocytes Prior to surgery, frogs were anesthetized by placing the animal in a 2 g/L solution of MS222 (3-aminobenzoic acid ethyl ester). Oocytes were removed from an incision made in the abdomen. To remove the follicu lar cell layer, harvested oocytes were treated with collagenase for 2 hours at room temperat ure in calcium-free ND96 solution (96 mM NaCl, 2 mM KCl, 1mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES, adjusted to pH 7.5 with 10N NaOH). Subsequently, stage 5-6 oocytes were isol ated and injected with ~25 ng of cRNA in 50 nl volume using a Micro4 inject or (WPI, Berlin, Germany). Oocytes were kept at 17C in sterile ND96 oocyte medium that was supplemented with 2.5 mM sodium pyruvate, 100 units per ml/1 penici llin, 100 mg per ml/1 streptomycin, and 5 % horse serum. Recordings were made three to seven days after injection.

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68 Oocytes injected with RNA produced from Construct 1 did not express a functional acHCN channel, i.e., no currents were generate d following hyperpolarizati on of oocytes injected with Construct 1 RNA. Therefor e, all the experiments describe d below were performed on the oocytes injected with Construct 2 RNA. 3.2.3 Voltage-Clamp Recording of the Whole-Oocyte Responses Oocytes were conditioned in the ND96 serum-free medium for 1 hr pr ior to recording and then placed in a small volume of bathing medi um in a plastic chamber which was continuously perfused at a rate of 2 ml/min. The chamber solution was connected to a virtual ground of a current monitor head stage (VG-2A-x100, Mo lecular Devices, Sunnyvale, CA) through two 2 mM KCl 2 % agar-bridged, Ag/AgCl reference electrode s (Figure 3-2). Microelectrodes (1-3 M ) were prepared with a P-2000 puller (Sutter Instrument Co., Novato, CA) from 1.2 mm borosilicate glass capillaries (WPI) and f illed with a solution used for patch clamp recording of the mouse HCN channel (mBCNG-1) expressed in X. laevis oocytes (Santoro et al., 1998): 5 mM NaCl, 107 mM KCl, and 10 mM HEPES, pH 7.5. The concentrations of Na+ and K+ in this solution are in the biolog ical range naturally occurring in X. laevis oocytes (Weber, 1999). Currents were measured with a two-elect rode voltage clamp (OC 725A, Warner, New Haven, Conn., USA) using a DigiData1200 acquisiti on system (Molecular Devices). Recordings were made at room temperature (~25C). All control experiments were done in ND96 solution. Oocytes were clamped at a holding potential of -30 mV. This potential was chosen because it was the mean resting potential of the oocytes injected with ac HCN RNA. Currents were allowed to stabilize for 10 min following electrode penetr ation. Oocytes were then hyperpolarized for 5 s from the -30 mV holding potential to more negative potentials ranging from -50 mV to -110 mV in steps of -10 mV. I did not hyperpolarize oocyt es further because at potentials negative to

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69 -110 mV induced currents became too large a nd damaged the cells. After each hyperpolarizing voltage step the currents were allowed to return to their base values for 5 s. To determine time constants, oocytes were hyperpol arized for 20 s from the -30 mV holding potential to more negative potentials ranging from -50 mV to -110 mV in steps of -10 mV. Current amplitudes were calculated by subtrac ting the current values at ~ 120 ms after the beginning of hyperpolarization (following relaxati on of capacitive currents), from the current values at 160 ms from the end of the hyperpol arizing voltage step, where the currents were stable. Tail currents were generated by hyperpolarizi ng oocytes from a holding potential of -30 mV to -110 mV for 2 s to activate the HCN channels and then stepped to -100 mV and in steps of 10 mV to -10 mV. Further depolarizat ion was not done, because at the potentials positive to 0 mV native outward currents (present in the control oocytes) developed, which changed the shapes of tail curr ents in the acHCN mRNA-injecte d oocytes. The amplitudes of tail currents were determined at 140 ms after th e beginning of the depolarizing step following relaxation of capacitive currents. Modulation of the acHCN channel by cAMP and cGMP was determined using membrane permeable analogs: 8-Br-cAMP and 8-Br-cGMP (sodium salts), because the CNBD of HCN channels is intracellular. After measuring contro l currents, oocytes were perfused with a solution of a cyclic nucleotide in ND96 for 10 min be fore recording. This time was chosen because acHCN-mediated currents did not significantly change following l onger perfusions with either 8-Br-cAMP or 8-Br-cGMP. Percent activation by cyclic nucleotides was calculated using the following formula: % activation = 100 100 I ImV 110 h cNMP mV, 110 h. To test the effect of inhibitors on acHCN, oocytes were perfused with a solution of either Cs+ or ZD7288 (Tocris, Ellisville, MO) in ND96 for 10 min and then washed for either 10 min or

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70 30 min following perfusion by Cs+ and ZD7288 respectively. In both cases, these were the minimal times required for the acHCN-mediated currents to return to their control values. Percent inhibition by Cs+ or ZD7288 was calculated at the potentials of their maximal inhibition of Ih, -110 mV and -90 mV respectively, using the following formulas: % inhibition by Cs+ = 100 I I 100Ctrl mV, 110 h mV 110 h and % inhibition by ZD7288 = 100 I I 100Ctrl mV, 90 h mV 90 h. Experiments were performed on several oocytes from at least two different frogs under each condition. Initial training for recordi ng channel activity from oocytes was done on the cnidarian (Physalia physalis) voltage-gated potassium channel cloned in Dr. Pe ter Andersons laboratory. This channel was chosen because of its high ra te of successful expression. The oocytes were injected with the channels RNA, incubated for 2 days in ND96 media and the currents generated in response to depolarizing voltage steps in vol tage-clamp configuration were recorded. This work was done in Dr. Andersons laboratory under the supervision of Rebecca Price, a technician in the laboratory who had exte nsive experience in r ecording from oocytes. A typical result of the experiments (n = 6) is shown in Figure C-1 of Appendix C. 3.2.4 Data Analysis Data were analyzed using ClampFit (Molecu lar Devices) and SigmaPlot 9.0 software (SYSTAT Software Inc., Point Richmond, CA, USA) Values depicted in graphs represent the mean s.e.m. from at least four independent experiments involving at least four different oocytes. 3.3 Results 3.3.1 Voltage Dependence and Kinetics The first and easiest step in determining wh ether the cloned acHCN channel is functional is to test its response to hype rpolarization. Stepping the memb rane potential from -30 mV to

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71 potentials negative to -70 mV in the acHCN RNA-injected oocytes produced slow inward currents characteristic of Ih (Figure 3-3A). These currents were not present in water-injected oocytes, n = 10 (Figure 3-3B). The amplitudes of the currents increased with the increase in activating voltage steps. The membrane potential of half-maximal activation (V1/2), determined by fitting the I/V curve with the Boltzmann equation, was -83.77 0.79 mV with a slope factor 7.23 0.70 mV, n = 4 (Figure 3-4). Upon return to the holding potential (-30 mV) the currents deactivated, generating decaying, inward tail currents. The time course of activation was strongly dependent on the hyperpolarizing voltage steps with values ranging from 6229.76 584.39 ms at -80 mV to 2330.06 156.62 ms at -110 mV, n = 18. Activation by hyperpolarization and slow kine tics of the recorded current support the prediction that the func tional form of the HCN channel was cloned. 3.3.2 Ion Permeability of acHCN A second major characteristic of a channel, in addition to the voltage dependence, is its permeability to different ions, reflected in the reversal potential (Er) of the channel. The reversal potential of acHCN was determined from the I/V curve for tail currents to be -25.84 1.19 mV, n = 17 (Figure 3-5). It was sh own in other organisms that HCN channels are permeable only to potassium and sodium ions (Robinson and Siegelbaum, 2003). Because the reversal potential of ac HCN is similar to those of other HCN channels, my hypothesis is that acHCN also is permeable only to K+ and Na+ with approximately the same ratio of permeabilities for these two ions. To test this hypothesis I changed the extracellular concentrations of Na+ and K+ from the control values (2 mM K+, 96 mM Na+) to 16 mM K+, 82 mM Na+ and 36 mM K+, 62 mM Na+. If the acHCN channel is permeable only to potassium and sodium ions then it is possible to predict

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72 how the reversal potential will change when I ch ange extracellular concentrations of these ions. Otherwise, if the predicted value of the re versal potential does not correspond to the experimentally determined one, it can be inferred that other io n(s) permeate the channel and participate in determining its reversal potential. In both cases, experimentally determined reversal potentials (-21.11 0.80 mV and -13.04 1.22 mV respectively) closely matched th e expected reversal po tential calculated for each oocyte (-20.84 1.41 mV and -14.47 0.97 mV respectively, n = 4) determined using the Goldman equation (Vm = F RT ln( n Na in K out Na out K]i [Na P ] [K P ] [Na P ] [K P ) based on the assumption that only these two ions contribute to HCN-mediated curre nt (Figure 3-6). This means that the acHCN channel conducts almost exclusiv ely potassium and sodium ions. Using the reversal potential and concentrations of sodium and potassium ions in extracellular and intracellular solutions, the relative permeability of acHCN to Na+ and K+ was determined by the Goldman equation to be 0.40. As with other HCN channels, elevating th e extracellular potassium concentration drastically increased amplitudes of Ih: 4.64 0.62 fold for [K+]ext = 16 mM and 7.82 0.30 fold for [K+]ext = 36 mM, n = 4 (Figure 3-7). The permeability of the cloned channel to both potassium and sodium ions and the dependence of Ih amplitude on extracellular potassium concentration confirm the identity of acHCN. 3.3.3 Activation of acHCN by Cyclic Nucleotides The third major characteristic of HCN channels is their activation by cyclic nucleotides. cAMP and cGMP activate the acHCN cha nnel by increasing the amplitude of Ih (Figures 3-8 and 3-9). 1 mM 8-Br-cAMP increased current amplit ude measured after stepping from -30 to

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73 -110 mV by 18.01 0.71 %, n = 4, and 1 mM 8-B r-cGMP increased current amplitude measured under the same conditions by 35.75 20.31 %, n = 4. The increase in the current amplitude by cycl ic nucleotides was concentration-dependent. The concentration of 8-Br-cGMP requi red for a half-maximal increase (Ka) was 2.16 0.68 M, n = 4 (Figure 3-10). Based on the obtain ed dose-response relationship, the Ka value for 8-BrcAMP is expected to be much larger. The exact concentration was not determined because at the maximal concentration of 8-Br-cAMP used (1 0 mM) the dose-response curve still did not plateau (Figure 3-11). An alternative expl anation is that the curve plateaus at 100 M 8-BrcAMP and the increase in Ih in the presence of higher concentr ations of 8-Br-cAMP is due to nonspecific effects. Unlike in most other cloned HCN channels, cycl ic nucleotides did not shift the voltage of the half-maximal activation of acHCN in a depol arized direction (-83.77 0.79 mV, slope factor 7.23 0.70 mV, n = 4, in control; -86.05 0.95 mV, slope 8.47 0.86, n = 4, in the presence of 1 mM 8-Br-cAMP, and -85.34 0.52 mV, slope 6. 93 0.46, n = 4, in the presence of 1 mM 8-Br-cGMP). The observed activation of acHCN by cyclic nucleotides also confirms that the clone used in experiments encodes a functional form of the channel. 3.3.4 Inhibition of acHCN by ZD7288 and Cs+ Finding inhibitors of acHCN is very important for studying the channel in A. californica neurons because blocking the acHCN-mediated curre nt will help to determine functional role(s) of the channel in both neuronal networks and behavior. As a part of the characterization of the cha nnel I first tested whethe r acHCN is inhibited by Cs+ in the millimolar concentrations shown to block other HCN channels (Glitsch et al., 1986). Replacing part of the extrace llular sodium with 2 mM Cs+ led to an almost complete block of

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74 acHCN-mediated currents (79.91 2.72% inhibi tion at -110 mV, n = 4) (Figure 3-12). The current amplitudes returned to their initial values following 10 min washout. Although Cs+ blocks HCN channels, it is not a specifi c inhibitor of the channels because it also blocks other classes of potassium channels (French and Shoukimas, 1985; Clay and Shlesinger, 1983). Thus, one cannot use Cs+ to specifically block HCN channels in A. californica neurons. Therefore, I also tested ZD7288, a spec ific inhibitor of HCN channels in mammals (BoSmith et al., 1993). ZD7288 inhibited the acHCN channel by decreasing the amplitude of Ih (Figure 3-13). 100 M ZD7288 decreased Ih by 83.86 .43 %, n = 4. This concentration of inhibitor was chosen because it is also used in vertebrates, such as guinea pigs and rats (Harris and Constanti, 1995; Kretschmannova et al., 2006). Inhibition of the acHCN channel by ZD7288 wa s concentration-dependent (Figure 3-14). The concentration of the blocker requ ired for a half-maximal inhibition (Ki) was 4.68 1.51 M, n = 4. ZD7288 did not change the voltage of the half-maximal act ivation of acHCN (-83.77 0.79 mV in the c ontrol and -83.93 3.66 mV in the presence of 100 M ZD7288, n = 4). Inhibition of the acHCN channel by Cs+ and ZD7288 additionally supports the identity and functionality of the channel. 3.4 Discussion 3.4.1. Voltage Dependence and Kinetics The acHCN channel was expressed in X. laevis oocytes. The properties of the channel, namely, its activation by both hyperpolarizati on and cyclic nucleotides, permeability to potassium and sodium ions and inhibition by Cs+ and ZD7288, confirm its identity as an HCN channel. This is one of the firs t successful expressions of any A. californica channel in oocytes (Pfaffinger et al., 1991; Quattrocki et al., 1994; Collado et al., 2007).

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75 Although acHCN displays the major biophysical and pharmacological properties of HCN channels, it has some unique features. Because the most significant difference between the HCN channels of A. californica and other organisms is in the N-te rminus, this region can potentially determine some of the differential properties of acHCN, such as its exceptionally slow activation kinetics (Table 3-1). Pascual et al. (1997) showed that the N-te rminal region of voltage-gated potassium channels effects their activation. Deletion of this region led to significantly slower activation times. Because HCN channels belong to a superg roup of voltage-gated potassium channels, the slow activation kinetics of acHCN may result from the truncati on of its N-terminal region (if translation of the channel starts from its first ATG codon). The mechanism of regulating ch annel activation by the N-term inal region in potassium voltage-gated channels may be charge transfer of the phosphorylated residues to a voltage sensor in TM4 (Perozo and Bezanilla, 1990). Because of the mutation of its first ATG codon, the acHCN channel might have lost both of its tw o potential N-terminal phosphorylation sites: by protein kinase C and casein kina se II (see Figure 2-2). Therefore, the suggested mechanism may be disrupted in the acHCN channel resulting in its slower kinetics. TM4 itself is almost identical in all HCN ch annels, including acHCN, but there are several other regions which may play an important role in determining activation kinetics: TM1 and linkers between TM1 and TM2 (Ishii et al., 2001 ), TM4 and TM5 (Chen et al., 2001) and TM6 and CNBD (Decher et al., 2004). TM1 and the TM1-TM2 linker of the vertebrate HCN1 (fast) and HCN4 (slow) channels differ in eight positions. Point mutation studies demonstrated that amino acids of the HCN4 channel in each of these eight positions contribute toward slower activation time constants (Ishii et al., 2001). In three of thes e positions the acHCN channel has the same amino acids as the

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76 HCN4 channel (L147, I153 and D165) which may cont ribute to its slow activ ation. In the other five positions studied by Ishii et al. (F143, V146, N164, D166 and L167), and five additional sites of TM1 and TM1-TM2 linker (L148, I149, A150, A158, S160) acHCN differs from both HCN1 and HCN4 (as well as HCN2 and HCN3). Ho wever, it is not known how these differences influence channel activation. The residues in the linker between TM4 and TM5 (in positions 279, 282 and 290 of the acHCN channel) and in the linker between TM 6 and CNBD (in positions 392, 394, 398, 400 and 404), shown to be crucial for gating other HC N channels, do not differ between acHCN and HCN channels cloned from both vertebrates and i nvertebrates, suggesting that these regions do not determine the slower activation kinetics of acHCN. The membrane potential of half-maximal activation of acHCN (-83.77 mV) is more positive than V1/2 of most other cloned invertebrate HCN channels. Still, it implies that at the potentials of typical after-hyperpolari zations following action potentials in A. californica neurons (-50-70 mV), only a small fraction of the acHCN channels are activated. However, in neurons, V1/2 of acHCN can be much more positive due to different lipid composition of oocyte membrane and A. californica neurons. It was shown that in cardiac and neuronal cells phosphatidylinositol-4,5-bisphosphate (PIP2) shifts voltage dependence of HCN channel activation by ~20 mV in a depol arizing direction (Zolles et al .; 2006; Pian et al., 2006). The voltage of half-maximal activation of acHCN may be more positive in A. californica neurons than in X. laevis oocytes due to lower levels of PIP2 in the membranes of the oocytes. Supporting this hypothesis, there is data th at phospholipase C, present in X. laevis oocytes, hydrolyzes PIP2 during maturation of oocytes (Jac ob et al., 1993; Han and Lee, 1995).

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77 In conclusion, although acHCN, like other inve rtebrate HCN channels, is activated by hyperpolarization, its very slow ki netics and rather depolarized V1/2 make it more similar to vertebrate HCN channels, especially HCN4 (see Table 3-1). 3.4.2 Ion Permeability of the acHCN Channel Because the pore region of the HCN channels of A. californica and other animals are very similar, I expected the permeability of acHCN to be comparable to that of the other HCN channels. Like them, acHCN conducts only Na+ and K+, and elevated extracellular potassium increases the amplitude of Ih by several-fold (Robinson and Siegelbaum, 2003). However, the relative Na+ / K+ permeability of acHCN (0.40) is higher than that of the HCN channels of most other animals (but lower than in fish HCN chan nels, e.g., 0.5 in goldfish; Tabata and Ishida, 1996). This makes the reversal potential of acHCN (-25.84 mV ) 4-12 mV more depolarized compared to other invertebrate HCN channels. Similar to V1/2, the Er value of acHCN is closer to that of vertebrate HCN ch annels (see Table 3-1). 3.4.3 Activation of the acHCN Ch annel by Cyclic Nucleotides As discussed in section 3.4.1, the voltage of the half-maximal activation of most invertebrate HCN channels ( 107-119 mV) lies outs ide of the physiologi cal potentials (see Table 3-1). Binding of the cy clic nucleotides shifts V1/2 by as much as 41 mV in a depolarizing direction (for P. argus HCN channel) thus moving it into a physiological range. As a result, cyclic nucleotides may activate invertebrate HC N channels and switch the activity mode of the HCN-expressing cells from sile nt to spiking. Although V1/2 of the acHCN channel is in the physiological range, cyclic nucleotides do not shif t it further in a depolarizing direction, but rather increase the amplitude of Ih. This effect may help the cells to recover faster from hyperpolarization following an action potential (A P) and proceed to firing another AP, thus

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78 increasing the frequency of disc harge. However, because cAMP does not significantly increase Ih at physiological potentials, this may be true only for cGMP. The difference between acHCN and both vertebra te and invertebrate HCN channels is that cGMP activates the channel to a larger extent than cAMP. Th ese results cannot be explained based on the available data about the structure-function relationshi p obtained from the vertebrate channels, since the residues shown to be im portant for cAMP binding (L526, R543, R584 and I588) are preserved in acHC N (Zagotta et al., 2003). The modulation of acHCN by cGMP also cannot be explained based on the data obtained from the vertebrate HCN channels. Zagotta et al (2003) showed that cG MP binds to the CNBD of the vertebrate HCN channels through the threonine in a po sition corresponding to amino acid 544 of acHCN and suggested that th e change of this threonine to valine eliminates activation of an HCN channel by cGMP as in the sea urch in HCN channel (Gauss et al., 1999). However, HCN channels of A. californica and insects ( D. melanogaster and A. mellifera ) also have valine in this position and they are activated by cGMP This implies that the mechanism of cGMP binding to the most invertebrate HCN channels ma y be different than vertebrate HCN channels. 3.4.3 Inhibition of the acHCN Channel by Cs+ and ZD7288 I showed that both Cs+ and ZD7288 inhibit the acHCN channe l in the concentrations used in other animals. Inhibition of acHCN by ZD7288 (83.86% by 100 M ZD7288) is similar to the inhibition of vertebrate HCN channels by th e same concentration of the bl ocker with the maximal percent of inhibition ranging from 84.3 % in the rat pi tuitary cells (Kretschmannova et al., 2006) to almost complete block in guinea pig substantia nigra neurons (Harris and Constanti, 1995), with the constant of half-maxim al inhibition being around 2 M (Harris and Constanti, 1995). In lobster, the only invertebrate animal wher e ZD7288 was tested, this blocker inhibited Ih almost

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79 completely at 100-300 M (Gisselmann et al., 2005b), but the concentration dependence of inhibition and, thus, the constant of inhibition were not determined. As in mammalian neurons (BoSmith et al ., 1993), ZD7288 inhibited the acHCN channel by decreasing the amplitude of Ih, but in contrast to its act ion in mammalian cells, ZD7288 did not change the voltage of the ha lf-maximal activation of acHCN. Inhibition by ZD7288 of HCN channels from the animals of all three superclades: Deuterostomia (mammals), Ecdysozoa (spiny lobster) and Lophotrochozoa ( A. californica ) suggests that this is a universa l inhibitor of the channels. A dditional evidence for that comes from a recent study by Cheng et al. (2007) showi ng that two residues in TM6 (Ala425 and Ile432 in the mouse HCN2 channel) are the primary de terminants for blocking the channels by ZD7288, and these residues are preserved among species (see Figure 2-2). 3.4.5 Summary Although major biophysical and pharmacological pr operties of acHCN are similar to those of other HCN channels, a number of characteri stics make it different from most other HCN channels. These characteristics include: very sl ow kinetics, activation by cyclic nucleotides by increasing Ih amplitude rather than shifting V1/2 in a depolarizing directi on, stronger activation of the channel by cGMP than cAMP, and the relatively depolarized reversal potential and potential of the half-maximal activation. Th e last two features suggest that the acHCN channel plays a role in controlling spiking frequency of A. californica neurons. Testing this hyp othesis, which is the subject of Chapter 5, was made easier by the finding that acHCN is inhibited by ZD7288, a specific blocker of the HCN channels. This inhi bitor allowed me to remove the HCN-mediated current and determine how it influe nces neuronal spiking. Howeve r, the specificity of ZD7288 in the intact A. californica brain and neurons still has to be confirme d to ensure that this blocker does not affect neuronal spiking of the cells th at do not express HCN ch annels. This issue is

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80 addressed in Chapter 5. Also, to study the role of acHCN in A. californica model neurons have to be chosen. To find these cells, I localized acH CN transcript in the CNS of the animal. These experiments are described in the following chapter.

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81 Figure 3-1. Schematic diagram of the constructs used to produce acHCN RNA. LingT plasmid is a modified BlueScript plasmi d with added 5and 3-UTRs of X. laevis -globin and T-overhangs. Two constructs made from a single transcript were cloned into the plasmid: Construct 1 starting from the al ternative start codon CTG and Construct 2 starting from the first ATG codon.

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82 Figure 3-2. Schematic diagram of a two-elec trode voltage clamp oocyte recording set-up. Oocytes were placed in a small volume of bathing medium in a plastic chamber which was continuously perfused at a rate of 2 ml/min. The chamber solution was connected through two 2 mM KC l 2 % agar-bridged, Ag/AgCl reference electrodes to a virtual ground of a current monitor head stage.

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83 Figure 3-3. Determination of the voltage depe ndence of the acHCN channel. Slow inward currents characteristic for HCN channels we re generated upon hyperpolarization from a holding potential of -30 mV to potentials negative to -70 mV (A). These currents were not present in water-injected oocytes (B). Ih amplitudes were determined as shown in A. N=10. 5 -80 -40 -100 0 0A B 5 0 -80 -40 -100 0Time, sTime, sIm, nAVm, mV Im, nAVm, mV Ih 5 -80 -40 -100 0 0A B 5 0 -80 -40 -100 0Time, sTime, sIm, nAVm, mV Im, nAVm, mV 5 -80 -40 -100 0 0A B 5 0 -80 -40 -100 0Time, sTime, sIm, nAVm, mV Im, nAVm, mV Ih

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84 Vm, mV -110-100-90-80-70-60I / Imax 0.0 0.2 0.4 0.6 0.8 1.0 Figure 3-4. Voltage dependence of the acHCN ch annel activation. The I/V relationship (solid line) of acHCN was determined by hyperpolarizing oocytes (n = 4) for 5 s from the -30 mV holding potential to more negative potentials rang ing from -60 mV to -110 mV in steps of -10 mV. V1/2 was determined by fitting the I/V curve with a curve calculated by the Boltzmann equa tion (dashed line) to be ~ -84 mV, n = 4.

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85 Figure 3-5. Determination of a reversal potenti al of the acHCN channe l. A) Tail currents (arrow) were generated by hyperpolarization of oocytes from a hol ding potential of -30 mV to -110 mV for 2 s to activate the HCN channels and then stepped to -100 mV and in steps of 10 mV to -10 mV. B) Larger magnification of the inward tail current at -110 mV, outward tail cu rrent at -10 mV a nd approximately zero current at -30 mV. C) Normalized amplitude s of the tail currents were plotted against voltage and the reversal pot ential of acHCN was determined at a point where the current changes its sign to be ~ -26 mV, n = 17. Time, sVm, mV Im, nA V m mV -100-80-60-40-20 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 -25.84 mV Time, sVm, mV Im, nAA B C-10 mV -30 mV -100 mV I I 0 I I / Imax Time, sVm, mV Im, nA V m mV -100-80-60-40-20 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 -25.84 mV Time, sVm, mV Im, nAA B C-10 mV -30 mV -100 mV I I 0 I I / Imax

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86 Figure 3-6. Dependence of the reversal poten tial of the acHCN cha nnel on extracellular potassium concentration. Experimentally de termined reversal potential (solid line) closely matched the reversal potential de termined using the Goldman equation based on the assumption that the acHCN channel is permeable only to K+ and Na+ (dashed line). Error bars in the calculated line reflec t that expected reversal potential in 16 and 36 mM K+ solutions were calculated based on the reversal potential in 2 mM K+ solution for each individual oocyte.

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87 Figure 3-7. Dependence of the Ih amplitude on extracellular potassium concentration. Elevating extracellular concentration of K+ drastically increases amplitudes of Ih: 4.64 0.62 fold for [K+]ext = 16mM (A) and 7.82 0.30 fold for [K+]ext = 36mM (B). Current traces produced by stepping from -30 to -110 mV in 2 mM extracellular potassium (control) are shown in black. Current trac es produced under the same conditions in extracellular solution with elevated K+ were superimposed with control traces and are shown in red. N = 4. 5 0 -100 -50 -400 -200 0 Time, sTime, sIm, nAVm, mV Im, nAVm, mV 5 0 -100 -50 -200 -100 0 A B Control Control 16 mMK+36 mMK+ 5 0 -100 -50 -400 -200 0 Time, sTime, sIm, nAVm, mV Im, nAVm, mV 5 0 -100 -50 -200 -100 0 A B 5 0 -100 -50 -400 -200 0 Time, sTime, sIm, nAVm, mV Im, nAVm, mV 5 0 -100 -50 -200 -100 0 5 0 -100 -50 -400 -200 0 Time, sTime, sIm, nAVm, mV Im, nAVm, mV 5 0 -100 -50 -200 -100 0 A B Control Control 16 mMK+36 mMK+

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88 Figure 3-8. Activation of the acHCN channel by cyclic AMP. A) Current trace generated by hyperpolarization from -30 to -110 mV in the presence of 1 mM 8-Br-cAMP (red) was superimposed on a current trace obtai ned under the same conditions in the absence of 8-Br-cAMP (black) in the bathing solution. B) I/V relationships in control conditions (black) and in the presence of 1 mM 8-Br-cAMP (red) The currents were normalized to the current value at -110 mV in the presence of 8-Br-cAMP. cAMP activates acHCN by increasing the Ih amplitude, with the maximal increase of ~ 18 % at -110 mV, n=4. However, cAMP does not change the acHCN-mediated current at the physiological potentials (-50-70 mV) and does not shift the voltage activation of the channel in a depolarizing direction. Vm, mV -110-100-90-80-70-60-50I / Imax 0.0 0.2 0.4 0.6 0.8 1.0 + 8-Br-cAMP Control 5 0 -100 -50 -100 -50 0 Time, sIm, nAVm, mVControl + 8-BrcAMPA B Vm, mV -110-100-90-80-70-60-50I / Imax 0.0 0.2 0.4 0.6 0.8 1.0 + 8-Br-cAMP Control 5 0 -100 -50 -100 -50 0 Time, sIm, nAVm, mVControl + 8-BrcAMPA B+ 8-Br-cAMP Control 5 0 -100 -50 -100 -50 0 Time, sIm, nAVm, mVControl + 8-BrcAMP 5 0 -100 -50 -100 -50 0 Time, sIm, nAVm, mVControl + 8-BrcAMPA B

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89 Figure 3-9. Activation of the acHCN channel by cyclic GMP. A) Current trace generated by hyperpolarization from -30 to -110 mV in the presence of 1 mM 8-Br-cGMP (red) was superimposed on a current trace obtai ned under the same conditions in the absence of 8-Br-cGMP (black) in the bathing solution. B) I/V relationships in control conditions (black) and in the presence of 1 mM 8-Br-cGMP (red) The currents were normalized to the current value at -110 mV in the presence of 8-Br-cGMP. cGMP is more potent than cAMP in activating acHCN with the maximal increase in the Ih amplitude of ~ 36 % at -110 mV, n=4. Vm, mV -110-100-90-80-70-60-50I / Imax 0.0 0.2 0.4 0.6 0.8 1.0 Im, nAVm, mV Time, sA BControl Control +8-BrcGMP +8-Br-cGMP 5 0 -100 -50 -100 -50 0 Vm, mV -110-100-90-80-70-60-50I / Imax 0.0 0.2 0.4 0.6 0.8 1.0 Im, nAVm, mV Time, sA BControl Control +8-BrcGMP +8-Br-cGMP 5 0 -100 -50 -100 -50 0 Time, sA BControl Control +8-BrcGMP +8-Br-cGMP 5 0 -100 -50 -100 -50 0

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90 8-Br-cGMP concentration, M 0.11101001000% Activation 0 10 20 30 40 50 Figure 3-10. Concentration dependence of acHCN activation by 8-Br-cGMP. Percent activation was calculated as an increase in Ih amplitude using the following formula: % activation = Ih-110mV, 8-Br-cGMP / Ih-110mV 100 100. Ka = 2.16 0.68 M. N = 4 for 0.1, 1 and 10 M 8-Br-cGMP, n = 6 for 100 M 8-Br-cGMP and n = 7 for 1000 M 8-Br-cGMP.

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91 Figure 3-11. Concentration dependence of acHCN activation by 8-Br-cAMP. Percent activation was calculated as an increase in Ih amplitude using the following formula: % activation = Ih-110mV, 8-Br-cAMP / Ih-110mV 100 100. Ka value for 8-Br-cAMP was not determined because at the maximal c oncentration of 8-Br-cAMP used (10 mM) the dose-response curve still di d not plateau. An alternativ e explanation is that the curve plateau at 100 M 8-Br-cAMP and the increase in Ih in the presence of higher concentrations of 8-Br-cAMP is due to unspecific effects. N = 4. 8-Br-cAMP concentration, M 110100100010000% Activation 0 10 20 30 40 50

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92 Figure 3-12. Inhibition of the acHCN channel by low concentrations of Cs+. A) Current traces generated upon hyperpolarization from 30 mV to -110 mV in a media with 2 mM Cs+ (red) or following washout (blue) we re superimposed on the current trace generated under the same conditions in a media without cesium (black). B) I/V relationships for acHCN in c ontrol conditions (black), in the presence of 2 mM of Cs+ (red) or following washout (blue). The curr ents were normalized to the current value at -110 mV under the control condi tions. 2 mM cesium decreased Ih amplitude by 79.91 2.72 %, n = 4. V m mV -110-100-90-80-70-60-50I / I max (Control) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Control Washout +Cs+(2mM) Time, s 5 0 -100 -50 0 -100 0 -50 Washout +Cs+(2mM) ControlIm, nAVm, mVA B V m mV -110-100-90-80-70-60-50I / I max (Control) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Control Washout +Cs+(2mM) Time, s 5 0 -100 -50 0 -100 0 -50 Washout +Cs+(2mM) Control Washout +Cs+(2mM) ControlIm, nAVm, mVA B

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93 Figure 3-13. Inhibition of acHCN by ZD7288. A) Current traces generated upon hyperpolarization from -30 mV to -90 mV in a media containing 100 M ZD7288 (red), or following washout (blue) were s uperimposed on the current trace recorded under the same conditions in the media without the inhibitor (black). B) I/V relationships in control conditions (black), in the presence of 100 M ZD7288 (red) or following washout (blue). The currents we re normalized to the current value at -110 mV under control conditions. 100 M ZD7288 decreased the amplitude of Ih by ~ 84 %. N = 4. V m mV -110-100-90-80-70-60-50 0.0 0.2 0.4 0.6 0.8 1.0 Washout Control ZD7288 Control Washout ZD7288 -100 -50 0 0 -50 5 0 Time, sIm, nA Vm, mVA BWashout Control ZD7288 Washout Control ZD7288 Washout ZD7288 -100 -50 0 0 -50 5 0 Time, sIm, nA Vm, mVA BI / Imax V m mV -110-100-90-80-70-60-50 0.0 0.2 0.4 0.6 0.8 1.0 Washout Control ZD7288 V m mV -110-100-90-80-70-60-50 0.0 0.2 0.4 0.6 0.8 1.0 V m mV -110-100-90-80-70-60-50 0.0 0.2 0.4 0.6 0.8 1.0 Washout Control ZD7288 Control Washout ZD7288 -100 -50 0 0 -50 5 0 Time, sIm, nA Vm, mVA BWashout Control ZD7288 Washout Control ZD7288 Washout ZD7288 -100 -50 0 0 -50 5 0 Time, sIm, nA Vm, mVA BI / Imax

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94 Figure 3-14. Concentration dependence of th e acHCN channel inhibition by ZD7288. Percent inhibition was calculated using the following formula: % Inhibition = 100 Ih-90 mV / Ih-90 mV, Ctrl 100. ZD7288 inhibits acHCN channel in a dose-dependent manner. Ki = 4.68 1.51 M, n = 4. ZD7288 concentration, M 0.1110100% Inhibition 0 20 40 60 80

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95Table 3-1. Characteristics a nd properties of HCN channels cl oned from different organisms Organism Channel name Protein length, aa Ion selectivity filter sequence Heterologous expression system Expression in native organism V1/2, mV Erev, mV Time constant, ms Shift by cAMP (1mM), mV K1/2, m Shift by cGMP (1mM), mV K1/2, m pK+/ pNa+ Function Reference HCN1 890 CIGYGA HEK293 -70 -21 67 Neonatal myocytes*, brain, heart -75 278 Rhythmic activity HCN2 889 CIGYGR HEK293 -93 97 -24 1961192 +15-16 5 HCN3 774 CIGYGQ HEK293 -77 -21 1244 Neonatal myocytes, brain, heart, testis -66 1271 Rhythmic activity Human HCN4 1203 CIGYGR HEK293 -82109 -22 26523000 +11-16 4.6 Ludwig et al., 1999; Qu et al., 2002; Seifert et al., 1999; Vaccari et al., 1999; Stieber et al., 2005 Sperm flagellum May control the waveform of flagellar beating SpHCN1 767 CIGYGK HEK293 -85 -30 0.74 5.6 Galindo et al., 2005 Sea urchin SpHCN2 638 SIGFGR Sperm flagellum Gauss et al., 1998 -116 5.66.3 Olfactory signal transduction Lobster PAIH 692 CIGYGS HEK293 -119 -36 297-1495 +41 17.6 +18 120 4.8 Gisselmann et al., 2005b Head, antennae (ORNs) body -107 230-355 Possibly olfactory signal transduction Honey bee AMIH 632 CIGYGR HEK293 -113 -37 50830 +19 5 +8 75 4.2 Gisselmann et al., 2003 Drosophila DMIH, Isoforms A1B2C2, A3B1C1, A2B1C1, A1B1C1 1319, 627, 618, 844 CIGYGR Antennae, brain, eyes, body Gisselmann et al., 2005a; Marx et al., 1999-2000 Aplysia californica acHCN 626 or 693 CIGYGR Xenopus oocytes CNS, aorta -84 -26 23306230 ? 2.16 2.5 Possible role in locomotion, feeding and memory Pavlo Kuzyk, unpublished *Tissues where HCN channels were studied

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96 CHAPTER 4 LOCALIZATION OF acHCN IN APLYSIA CALIFORNICA 4.1 Introduction In Chapter 3, I characterized the biophysical and pharmacological properties of the acHCN channel. Finding that ZD7288, a specific bloc ker of HCN channels, inhibits acHCN allows removal of HCN-mediated currents and determina tion of the functional role of the channel in A. californica However, most transcripts ar e expressed only in specific Aplysia neurons. Thus, the major objective for this chapter was to localize the acHCN transcript in the A. californica CNS and to identify neurons related to defi ned neuronal networks. This mapping permitted a study of the role of the channels in these ce lls. Specifically, because HCN channels depolarize neurons toward firing action poten tials, I determined the role of acHCN in controlling the frequency of the neuronal spiking (see Chapter 5) One condition that produces an increase in the frequency of the neuronal spiki ng is nerve injury. A prolonged incr ease in spiking frequency is the major manifestation of the LTH, the cellular analog of neuropa thic pain (Weragoda et al., 2004). Because HCN channels were s uggested to play an important role in nerve injury-mediated responses (Chaplan et al., 2003), I also determ ined how the expression of the acHCN mRNA changes following nerve injury. In other organisms HCN channels have been f ound mostly in sensory cells, such as rod and cone photoreceptors in the tiger salamander (Bader et al., 1979; Hestrin, 1987), newt (Satoh and Yamada, 2002) and lizard (Maricq and Korenbr ot, 1990); in B-type photoreceptors in the mollusk Hermissenda crassicornis (Yamoah et al., 1998); in the compound eye and chemosensory organs of D. melanogaster (Marx et al., 1999-2000); in the antennae of budworm, H. virescens (Krieger et al., 1999); in the h ead and antennae of the honey bee, A. mellifera (Gisselmann et al., 2003); and in the stretc h receptor neuron (Edman and Grampp, 1989) and

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97 olfactory receptor neur ons of spiny lobster, P. argus (Gisselmann et al., 2 005). In these cells HCN channels play a modulatory role. HCN channels expressed in interand motoneur ons often control rhythmic bursting of the neurons in central pattern generators, thus determ ining repeated activities, such as heartbeat in the medicinal leech, H. medicinalis (Angstadt and Calabrese, 1989), contraction of stomach muscles in lobster (Zhang et al., 2003) and rhythmic ingestion movements in L. stagnalis (Straub and Benjamin, 2001). In addition to the nervous system, HCN channe ls are also expressed in the hearts of vertebrates (Noma and Irisawa, 1976; Goto et al ., 1985; Baker et al., 1997) with the strongest expression in the sinoatrial node, the main pacemaker of the heart, where they contribute to the generation of the rhythmic activity. They are also found in the flagella of sperm of the echinoderm S. purpuratus (Gauss et al., 1998) and humans (Sei fert et al., 1999) where they are proposed to control the waveform of spermatozoan flagellar beating; in th e salivary gland cells of the giant Amazon leech, Haementeria ghilianii (Wuttke and Berry, 1992) and in rat pancreatic -cells (El-Kholy et al., 2007). E xpression of HCN channels in other tissues, e.g., liver, lung, kidney and skeletal muscle were found by some investigators (Santoro et al., 1998), but not others (Ludwig et al., 1998, 1999). To the best of my knowledge, no HCN channel expression has been demonstrated in the he art of an invertebrate animal. Because many neurons and neuronal networks in A. californica are identified, it is possible to determine the expression of acHCN in the CNS of the animal on the level of not just organs and tissues, but individual cells And since the role of many ne urons and neuronal networks in controlling different behaviors in Aplysia is known, localizing acHCN in specific cells will allow predictions about the function of the channel to be made and will identify candidate cells for functional studies.

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98 4.2 Methods 4.2.1 Animals A. californica (60-300 g) were obtained from Ma rinus (Long Beach, CA) or the NIH Aplysia Resource Facility (Miami, FL) and kept in tanks with natural sea water at 15-18C on a 12:12-hour light: dark cycle for up to 3 weeks. Before dissection, animals were anesthetized and killed by injection of 60 % ( volume/body weight) isotonic MgCl2 (337 mM). 4.2.2 Surgery An individual A. californica was weighed, anesthetized by injection of 35 % (volume/body weight) of isotonic MgCl2 (337 mM) and placed into the special chamber with sea water. The animal was fixed with hooks so that its dorsal surface was abov e water level to avoid contamination by bacteria, and a small cut was ma de to open the central ganglia (Figure 4-1). Then all nerves of the pedal ganglion on one or both sides were crushed, using #4 forceps, by applying firm pressure 2 cm from the ganglion. The incision was sutured and animals returned to the aquaria. Five days after surgery, the CNS of the animals was dissected and in situ hybridization performed using a probe to acHCN. This time was chosen because the strongest response to nerve injury, manifested in the incr ease in neuronal spiking, develops in 5 days following nerve crush (Lewin and Walters, 1999). 4.2.3 Whole-Mount i n situ Hybridization Protocol pCR4-TOPO plasmid containi ng cDNA fragments of intere st was linearized with appropriate restriction enzymes (Not I and Pme I in the case of acHCN). Transcription with T3and T7 polymerases (Roche Diagnostics, Basel, Switzerland) was done to obtain antisense and sense (control) RNA probes respectively, followi ng the Roche protocol for probe preparation with a DIG RNA labeling kit. RNA probes to acHCN were made to the first 1.5 kb of the channels cDNA.

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99 The in situ hybridization protocol (J ezzini et al., 2005) was modi fied from Bogdanov et al. (1996) and Ono et al. (2000). This method is relatively sensitive as it allows detection of low abundance transcripts, such as A. californica two-pore potassium channel AcK2p1 (Jezzini and Moroz, 2004) and fasciclin (Bastia ni et al., 1987). Briefly, after dissection, the CNS was treated at 34C for 30-50 min (depending on the size of an animal) with 10 g/ml protease IX, fixed for 3-6 hours with 4 % paraformaldehyde in phosphate buffer solution (PBS, 0.1 M, pH=7.4) and desheathed. Desheathed ganglia were dehydrated by subsequent 10 min incubations in 30 %, 50 %, and 70 % methanol in PTW (0.1 % Tween 20 in PBS), then in 100 % methanol for 5 min. After re-hydration, the ganglia were treated with 10 g/ml proteinase K (Roche) in PTW at room temperature for 1 hr, followed by post-fixation fo r 20 min at +4 C in 4 % paraformaldehyde and subsequent washes in 2 mg/ml glycine in PTW (2 times) and in PTW alone (2 times). Preparations were washed two times in 0.1 M tr iethanolamine hydrochloride, pH 8.0 (TEA HCl) and incubated in 2.5 l/ml solution of acetic an hydride in TEA HCl for 5 min; then 2.5 l/ml acetic anhydride was added, followed by 5 min inc ubation with agitation. Af ter several washes in PTW, the ganglia were incubated in hybridi zation buffer (50 % formamide, 5 mM EDTA (Invitrogen), 5X SSC, 1X Denhardt solution (0.0 2 % ficoll, 0.02 % polyvinylpyrrolidone, 0.02 % BSA), 0.1 % Tween 20, 0.5 mg/ml yeast tR NA (Invitrogen)) at 50C for 6-8 hrs. 0.5-2 l of probe (0.5-0.8 g/ l) was then added and hybridiz ation proceeded at +50C for 12 hr, followed by subsequent washes in string ent conditions (50 % formamide/ 5X SSC/ 1 % SDS (USB Corp., Cleveland, OH), then 50 % fo rmamide/ 2X SSC/ 1 % SDS, and then 0.2X SSC, twice, for 30 min at +60C each). Immunological detection was performed usi ng components of the DIG Nucleic Acid Detection Kit (Roche). Devel opment proceeded until background stai ning started to appear (1020 min in the case of serotonin transporter, 1.53 hrs for HCN and 2-4 hrs for CNG transcripts).

PAGE 100

100 Ganglia were then fixed in 4 % paraformaldehyde in methanol for 1 hr and washed in 100 % ethanol two times for 10 min. A schematic diagram of the procedure is shown in Figure 4-2. 4.2.4 Imaging Images were taken with a Digital Sight camera (Nikon, Melville, NY) mounted on an upright SZX12 microscope (Olymp us, Center Valley, PA). Figur es were prepared using Adobe Photoshop Elements 4.0 and Corel Draw 11. 4.2.5 Densitometry and Cell Counts Densitometry and cell counts were perfor med on pedal ganglia stained for acHCN, tryptophane hydroxylase or serot onin transporter on a Nikon Eclip se TE2000 microscope using MetaMorphTM program (Universal Imaging Corp ., Downingtown, PA). Brightness of neurons was determined and then converted to the intens ity of staining. Cells were considered to be stained if their intens ity of staining was two or more times higher than darkest background staining. Intensity of staining of the neurons was determined at the focal plane where they appeared darkest. A region was considered to be background if there wa s only monotone staining (if any) of neurons and interspersed glia. Gr aphical output and statis tical data have been generated using SigmaPlot. 4.3 Results 4.3.1 Localization of acHCN RNA in A. californica Using whole-mount in situ hybridization (n = 16) I have determined the distribution of acHCN transcripts in the CNS of A. californica (Figure 4-3, 4-4). As for most Aplysia transcripts, this distribution is cell-specific, i. e., only a subset of ne urons expresses the HCN mRNA. In contrast to other organi sms, known sensory neurons of A. californica do not express detectable amounts of HCN channels (Figure 4-3, 4-4). The stronge st staining for acHCN

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101 transcript was in motoneurons of buccal ganglia (20-25 cells, 20-110 m in diameter) (Figure 43A, 4-4, Table 4-1), metacerebral cells (MCC), 812 cells in E-clusters (10-40 m in diameter) and eight-nine cells in G-clusters (10-50 m) in cerebral ganglion (Figure 4-3B, 4-4, Table 4-1), which are part of feeding network, and up to 100 labeled cells (5-110 m in diameter) in pedal ganglia (Figure 4-3C, 4-4, Tabl e 4-1), with the highest level of expression between the pedal commissure and the pedal-pleural connective, wh ere locomotory neurons are located (Hening et al., 1979). The number of cells stained in the abdominal ganglion was variable. The most consistently stained cells were found in the le ft dorsal semi-ganglion, includ ing two big cells approximately 130 and 200 m in diameter, presumably L7 and L 11. The relative staining of these two cells differed among preparations. Two to six cells stai ned on the lateral surface of the left side and one to three cells on the medial caudal part of the left side on the ventral surface of the abdominal ganglion. There also was staining in 10 -20 cells in the medial and posterior clusters (5-60 m) and two cells in the anterior clus ter of the left pleural ganglion (40-50 m) (Figure 43D, 4-4, Table 4-1). Also, I found acHCN staining in the aorta, but not in the heart of A. californica (Figure 45). The staining in other organs, including ki dney, esophagus, salivary glands, gonads, gill, buccal mass, rhinophore and visceral complex was not significantly highe r than the background. These experiments were done on stage 11-12 animals, n = 4. Control experiments using sense probes and identical labeling pr otocols and conditions (n=6) did not produce specific staining in the CNS. 4.3.2 Down-Regulation of the acHCN Transcript Following Nerve Injury As discussed in Chapter 1, HCN channels are proposed to play a role in the LTH following nerve injury, manifested in increased spiking fr equency of neurons (Chapl an et al., 2003). Based

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102 on this finding, I hypothesize that expression of the acHCN transc ript increases following nerve injury. To test this hypothesis, I crushed all the nerves of one of the paired pedal ganglia and performed in situ hybridization five days following the ne rve crush using a pr obe to acHCN. Contrary to my prediction, acHCN mRNA is significantly down-regulated following nerve injury as demonstrated by the de crease in both the number of stained cells (30.67 10.07 vs 69.67 13.05, p = 0.0147, n = 3; Figure 4-6A, B) and intensity of staining (76.72 11.31 vs 115.61 14.52 arbitrary units, p < 10 e-10, n = 3; Figure 4-6A, C) in the pedal ganglia with the crushed nerves as compared with the identica l cells in the contralateral ganglia (control). Many pedal neurons, in which expression of the acHCN mRNA changes following nerve injury, are serotonergic (Hernadi et al., 1992, also see staining fo r serotonin transporter in Figure 4-8). In vertebrates, the concentr ation of serotonin (5-HT) increases at the site of nerve injury (Sharma et al., 1990; Salzman et al., 1987). Sero tonin activates HCN ch annels through its stimulation of cAMP production (Pape and McCormick, 1989). cAMP, one of the main transcription regulators (Barbas et al., 2003) may, at the same time, down-regulate expression of acHCN mRNA, thus preventing excessive exci tability of neurons. Because of these considerations, I decided to test how the tran scripts involved in serotonin metabolism change their expression following nerve injury in A. californica Two processes contribute to the increase in se rotonin concentration: serotonin release and serotonin uptake. Therefore, for this study I chose two transcripts that play major roles in these processes, specifically, tryptophane hydroxylas e (gi:94434834), the main enzyme in the synthesis of 5-HT, and serotonin transporter, res ponsible for 5-HT uptake. These transcripts have been cloned in the Moroz laborator y earlier (Moroz et al., 2006) Tryptophane hydroxylase mRNA is significantly down-regulated following nerve injury in the pedal ganglia with both in tact and crushed nerves (from 116 12.73 stained cells to 53

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103 8.49 and 25 4.24, p = 0.01 and 0.03 respectively, n = 2, Figure 4-7). Expression of serotonin transporter RNA also decreased following nerve in jury, and only in the pedal ganglia ipsilateral to nerve crush (31 8 stained cells vs 86 5 in contralateral ganglia, p = 7 e-10, n = 10; Figure 4-8B, 4-9A). There was no signifi cant difference between the nu mber of neurons stained for serotonin transporter in left and right ganglia in control preparations (Figure 4-9B). In addition to HCN, I cloned another channe l activated by cyclic nucleotides, cyclic nucleotide-gated (CNG) channel, and determined its localization in the CNS of A. californica The results of these experiment s are described in Appendix D. 4.4 Discussion 4.4.1 Localization of the acHCN RNA in A. californica As is the case with other HCN channels, acHCN is most abundantly expressed in the nervous system. The biggest surprise of this stud y was the lack of the transcript expression in sensory cells. (However, because localization of HCN channels was studied in detail in species of only two phyla (Chordata and Arthropoda), this may also hold true for animals of the other 32 phyla). Instead, the acHCN transcript is expresse d in motoneurons of the buccal, cerebral, pedal and abdominal ganglia. The motor output of many of these cells is known. For example, buccal motoneuron B3 innervates intrinsic buccal muscle 3 (I3) that participates in ingestive buccal movements (Church and Lloyd, 1994; Keating a nd Lloyd, 1999). Because B3 is one of the largest buccal motoneurons and one of the easiest to identify, it was chosen as a target neuron to study the functional role of acHCN. The cells of cerebral Eand G-clusters, along with buccal motoneurons, are a part of the feeding network controlling buccal muscles respons ible for protraction and retraction of the buccal mass during feeding (Jahan-Parwar and Fr edman, 1983; Jing and Weiss, 2001). In these cells acHCN channels may play a similar role as in L. stagnalis determining membrane potential

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104 and bursting properties of the cells of a central pattern generator controll ing rhythmic ingestion movements (Straub and Benjamin, 2001). MCCs are a bilateral pair of large serotonergic neurons (Weiss and Kupfermann, 1976). They are among the largest cells of the A. californica CNS, they can be easily visually identified and their function is known. The MCCs act centra lly to accelerate or trigger bursting in buccal motoneurons, including B3 neuron (Fox and Lloyd, 1998). They also act peripherally, modulating muscle contractions, and contributing to an arousa l state induced by food stimuli (Weiss et al., 1978). Specifically, they enhance the speed and strength of biting. Spiking frequency of MCCs correlates w ith the strength of ingestive movements (Rosen et al., 1989). Therefore, I used these neurons as anot her model to study the role of acHCN. In abdominal ganglia, the most consistently la beled neurons are likely to be beating cells L7, L8, L91 and L92 and bursting cell L11. acHCN may dete rmine their spontaneous activity (for a mechanism, see Chapter 1). L7, L91 and L92 cells control gill contraction and are a part of the defensive network. The large and well-charact erized L7 neuron was extensively used in memory-related studies (Kandel, 19 76) and is a potential candidate for future study of the role of the acHCN channel in LTP. In pedal ganglia, the acHCN channel is most strongly expressed in the pedal locomotory neurons (Hening et al., 1979) and it is likely, given the crucial role of the channel in controlling rhythmic movements in other animals, that in A. californica the HCN channel may control locomotion. Pedal locomotory neur ons are relatively large (80-150 m in diameter) and four of them, P1 through P4, are identifiable. P4 neuron is the easiest to identify due to its position and size. P4 is the largest ne uron (with a size of 80-120 m) in the region of th e dorsal side of pedal ganglia near the pedal commissure (Hening et al ., 1979). Therefore, this neuron was chosen as another model for study of the functiona l role of acHCN. Also, because in A. californica pedal

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105 ganglia are used to study the e ffects of nerve injury on transcri pt expression, it is possible to determine how expression of acHCN changes fo llowing nerve injury in pedal locomotory neurons. 4.4.2 Down-Regulation of the acHCN Transcript Following Nerve Injury As described in section 4.3.2, my hypothe sis about up-regulation of acHCN transcript following nerve injury was not confirmed. Expr ession of the HCN mRNA following nerve injury also decreases in the rat dor sal root ganglia (Chaplan et al., 2003). However, the Ih density in this system increases after nerve liga tion. It is possible that in A. californica the HCN protein level also increases following nerve injury and thus the Ih amplitude increases too. In this case, downregulation of the acHCN mRNA could prevent ex cessive neuronal excitability. To test this possibility, immunohistochemistry using antibodies to acHCN has to be performed, and the amplitude and voltage-dependence of Ih have to be measured in control preparations and in ganglia with crushed nerves. Expression of many A. californica transcripts increases following nerve injury, e.g., tolloid (Lovell et al., 2005), non-coding RNAs (Bodnarova and Moroz, unpublished observations), etc. This suggests that down-regulation of the acHCN mRNA was not due to a nonspecific reduction of RNAs following nerve injury. Down-regulation of the serotonin transporter transcript is expected to increase 5-HT concentration around serotonergic cells (due to d ecreased clearance) and t hus induce synthesis of greater quantities of cAMP. T hough at physiological potentials cAMP activates the HCN of A. californica only to a minimal extent, it may be re sponsible for the do wn-regulation of the acHCN mRNA. To determine a role of serotoni n in regulation of the acHCN transcript expression (through cAMP), experiments with i nhibitors of each stage in the cascade 5-HT cAMP acHCN have to be performed.

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106 4.4.3 Summary Localization of acHCN to mo toneurons of the buccal, cerebral, abdominal and pedal ganglia that are part of feeding, defensive and locomotory networks suggests a potential role for the channel in controlling the associated behaviors. Change in expression of acHCN transcripts following nerve injury implies that the channel ma y also play a role in neuronal responses to nerve injury. However, to clarify what that role is, studies on the level of proteins have to be performed. Giant interneuron MCC in the cerebral ganglion, buccal motoneuron B3 and pedal locomotory neuron P4 were chosen as the models to study a functional role of the HCN channels, which is a subject of the following chapter.

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107 Figure 4-1. Surgery design for pedal nerve crush. A. californica is anesthetized, fixed with hooks, and a small incision made in the front dorsal part of the animal, through which the nerves are crushed. The incision is then closed and the animal returned to the tank.

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108 Figure 4-2. Schematic diagram of in situ hybridization. RNA labeled with DIG (dioxigenin) probe is hybridized with the complementar y mRNA of interest; anti-DIG antibodies (Ab), coupled to alkaline phosphatase (A P), and NBT/BCIP (Nitr o-Blue Tetrazolium / 5-Bromo-4-Chloro-3'-Indolyphosphate p-Tolu idine, chromogenic substrate of AP) are added. AP reacts with NBT/BCIP solution and turns it purple thus indicating the presence of the mRNA of interest.

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109 Figure 4-3. Expression of the acHCN channel transcript in the CNS of A. californica as determined by in situ hybridization. The stronge st staining is in the buccal motoneurons and Eand G-clusters of th e cerebral ganglion (which are part of a feeding network), pedal locomotory neur ons and several individual cells in the abdominal ganglion, some of which may be important in learning and memory. A) Buccal ganglia. SC-sensory clusters. B) Cere bral ganglion. C) Le ft pedal and pleural ganglia. D) Abdominal gang lion. All scale bars = 500 m C D SC E cluster G cluster R2 L7L11 Pedal locomotory neurons R3R13 Left Right A B

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110 Figure 4-4. Schematic diagram of the expression of the acHCN transcript.

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111 Figure 4-5. Expression of acHCN in the aorta of A. californica as determined by in situ hybridization. A) Heart (H ) and aorta (A). The heart wa s cut in two to show its internal structure. Scale bar = 2 mm. B) Cl ose-up view of aorta. Scale bar = 1 mm. A BH A A BH A

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112 Figure 4-6. acHCN mRNA expressi on is decreased following nerve injury. A) Staining of pedal ganglia by i n situ hybridization using a probe to acHCN. Scale bar = 1 mm. B) Number of the stained cells is decreased in the pedal ganglia with crushed nerves five days after nerve injury (p = 0.0147, n = 3). C) Intensity of staining is decreased in the pedal ganglia with crushed nerves 5 days after nerv e injury (p < 10 e-10, n = 3). A.U.Arbitrary units

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113 Figure 4-7. Expression of tr yptophan hydroxylase mRNA is decreased following nerve injury in pedal ganglia both ipsiand contralateral to the nerve crush. A) Expression of tryptophan hydroxylase mRNA in contro l pedal ganglia as determined by in situ hybridization. Scale bars in A and B = 500 m. B) Expression of tryptophan hydroxylase mRNA in pedal ganglia after al l nerves of the right gaglion were crushed. C) Number of ne urons stained for tryptophane hydroxylase decreased from 116 12.73 in control to 53 8.49 and 25 4.24 in pedal ganglia contraand ipsilateral to the nerve crush p = 0.01 and 0.03 respectively, n = 2).

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114 Figure 4-8. Expression of sero tonin transporter mRNA in the A. californica CNS as determined by in situ hybridization. A probe to the se rotonin transporter mRNA labels many known serotonergic neurons, including met acerebral cells (MCC), neurons of CG and CC clusters of cerebral ganglion, several cells in the right rostral part of the abdominal ganglion, neurons around the pedal commisure and on the caudal surface of pedal ganglia. A) Cerebral ganglion. B) Pedal and pleural ganglia. All nerves of the left pedal ganglion were crushed while ne rves of the right peda l ganglion were left intact. C) Abdominal ganglion. D) Buccal ganglia. E) Staining of pedal ganglia with a sense probe.

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115 Figure 4-9. Expression of serot onin transporter mRNA is decreased following nerve injury. A) Number of stained cells is decreased in th e pedal ganglia with crushed nerves five days after nerve injury (p = 7e-10, n = 10). B) Numb er of neurons stained for serotonin transporter is not significantly di fferent between left and right ganglia in control preparations (n = 5).

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116 Table 4-1. Expression of th e acHCN mRNA in the CNS of A. californica Ganglia and clusters Number of stained neurons Size of stained neurons, m Identifiable neurons Neuronal network Reference Buccal 20-25 20-110 B3, B4, B5, etc Feeding Church and Lloyd, 1994 Cerebral E cluster G cluster 8-12 8-9 10-40 10-50 MCC Feeding Weiss and Kupfermann, 1976; Jahan-Parwar and Fredman, 1983 Pedal Up to 100 5-110 P1-P4 Locomotory Hening et al., 1979 Pleural 10-22 5-60 ? Abdominal 16-30 5-200 L7, L11 Defensive Kandel, 1976

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117 CHAPTER 5 CHARACTERIZATION OF THE FUNCTIONAL ROLE OF acHCN 5.1 Introduction The expression of acHCN in X. laevis oocytes and biophysical and pharmacological characterization of the channel, described in Ch apter 3, confirmed that the cloned channel is functional. Knowing the proper ties of acHCN and confirming th at ZD7288 blocks the channel allowed me to study the role of acHCN in A. californica neurons. Thus, the major objective of the work presented in this chapter was to determine the functional role of acHCN in Aplysia neurons. There are two goals for this chapter: first, to determine the role of acHCN in spiking of individual neurons and second, to determine th e contribution of acHCN in the maintenance of the feeding and locomotion rhythms. As discussed in Chapter 1, HCN channels depolarize cells and drive them towards the threshold for firing action potenti als. Therefore, the initial hypot hesis was that acHCN controls the spiking frequency of neurons that expre ss the channel and that blocking acHCN would decrease the frequency of the neuronal spiking. By localizing the acHCN transcript in the CNS of A. californica described in Chapter 4, target neurons for this study have been selected i.e., MCCs, arousal inte rneurons, which activate buccal feeding motoneurons (Weiss and Kupfer mann, 1976), buccal motoneuron B3, which controls jaw movement during feeding (Gardne r, 1971) and pedal locomotory motoneuron P4, which controls foot muscle contraction du ring locomotion (Hening et al., 1979). I used synaptically-isolated MCCs to determine the role of acHCN in controlli ng the spiking frequency on the level of individual neurons because MCCs are the most easily identifiable neurons expressing the acHCN transcript. To establish the role of acHCN on the systemic level, I determined the role of acHCN in controlling the spiking frequency of B3 and P4 ne urons, discharges of which directly correlate

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118 with the contraction of buccal and pedal muscle s, respectively, associated with feeding and locomotion, correspondingly (Church and Ll oyd, 1994; Hening et al., 1979). Thus, demonstrating how acHCN effects spiking of these neurons will allow predictions to be made about the role of acHCN in th e above mentioned behaviors. 5.2 Methods 5.2.1 Electrophysiological Recording from A. californica Neurons Animals were dissected as described in secti on 4.2.1. Treatment time of the isolated CNSs with protease IX (10 g/ml) was decreased to 20 min to avoid damage to the neurons. Because neurons of the abdominal ganglion were most sensitive and ruptured even following very short digestion times, I did not treat them with protease. The CNS was then desheathed with fine forceps and scissors to expose neuronal somata. The preparation was put in a sylgard-covere d Petri dish (3.5 cm in diameter) and continuously perfused with artificial Aplysia saline (AS, 460 mM NaCl, 10 mM KCl, 11 mM CaCl2, 55 mM MgCl2, 10 mM HEPES, pH 7.6-7.7, adjusted with 10N NaOH). To determine the role of acHCN at the le vel of individual neurons, I perfused the preparation with high divalent catio n (Hi-Di) solution, containing 1.25x Ca2+ and 2.2x Mg2+ compared to AS. This solution blocks synaptic transmission between ne urons and thus allows one to synaptically isolate them (Liao and Walters, 2002). Sharp intracellular electrodes (15-20 M ) were drawn with a P-2000 puller (Sutter Instrument Co) and filled with 3 M potassium acetate. All recordings were done using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA). Signals were digitized using a Digidata 1320A analog to digital converter, and analyzed using pClamp software (all from Axon Instruments).

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119 The R15 neuron (Halstead and Jacklet, 1974) wa s used to determine the specificity of ZD7288 for acHCN because no acHCN expre ssion was detected in this neuron by in situ hybridization. R15 was identified by its medial position in the right caudal quarter-ganglion of the abdominal ganglion, its large size and spont aneous spiking. B3 and P4 neurons were identified based on their position and size (see Chap ter 4). Spontaneous spiking was recorded for R15 and P4 neurons and spiking initiated by inje cting depolarizing current, typically 2 nA, was recorded for MCCs and B3 because these neur ons are usually silent except during feeding (Weiss and Kupfermann, 1976; Fox and Lloyd, 1998) Following penetration of neurons with electrodes, neuronal spiking was a llowed to stabilize for ~ 30 min, so that spontaneous spiking or spiking in response to the positive current inje ction was regular for at least 10 min. Control spiking was then recorded. Spiking was reco rded again following 1 hr perfusion of the preparation with AS or Hi -Di solution containing 150 M ZD7288. This concentration of the inhibitor was chosen because it causes maxima l inhibition of the heterologously-expressed channel (see section 3.3.4). All electrophysiological reco rdings were performed at room temperature (20-22C). 5.2.2 Data Analysis Data were analyzed using ClampFit (Molecu lar Devices) and SigmaPlot 9.0 software (SYSTAT Software Inc). Values depicted in graphs represent the mean s.e.m. 5.3 Results 5.3.1 Specificity of ZD7288 in A. californica Because ZD7288 has never been tested in mollusks I verified the specificity of this blocker for acHCN by determining whether ZD7288 changes spiking of the identifiable A. californica neurons that do not show detectable expression of the channel with our in situ hybridization. By localizing the acHCN tran script in the CNS of A. californica two potential targets for this study

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120 have been selected, both in the abdominal ga nglion: osmoregulatory neurons R3-R13 (Gainer and Wollberg, 1974) and R15 neuron controlling re spiratory pumping (Alevizos et al., 1991). To test the specificity of ZD7288 I se lected the R15 neuron, which is large and easil y identifiable. ZD7288 (150 M) did not significantly change patte rns of electrical activity of R15 spiking, including the number of spikes per minut e (1.19 0.40 fold change following perfusion with AS containing ZD7288, compared to control, p = 0.68, n = 3, Figure 5-1A, B, C), spike amplitude (1.01 0.01 fold change, p = 0.64, n = 3, Figure 5-1A, B, D) or maximal hyperpolarization (0.95 0.06 fold change, p = 0.51, n = 3, Figure 5-1A, B, E). These results suggest that ZD7288 is indeed a sp ecific inhibitor of acHCN and can be used to determine the role of the channel in neuronal spiking. 5.3.2 Effect of ZD7288 on the Spiking Frequenc y of Individual Neurons Expressing acHCN To determine the role of acHCN in contro lling the frequency of neuronal spiking, I blocked the channels by perfus ing the preparation with ZD 7288. Confirming my hypothesis, stated in section 5.1, ZD7288 significantly decr eased spiking frequency of MCCs (70 5.03 % decrease following perfusion with AS containing ZD7288, compared to control, p = 0.005, n = 3, Figure 5-2). Following washout, the spiking frequency recovered very slowly, with a recovery of 26.50 8.50 % of control value following 2 hr washout (n = 3) and 58 % of control value following 3 hr washout (n = 1). To ensure that decreased spiking of MCCs was not due to a decrease in synaptic input from other acHCN-expressing neurons, MCCs were synaptically isolat ed by perfusing the preparation with Hi-Di solution. Similar to its effect in AS, ZD7288 significan tly decreased spiking frequency of the synaptically-i solated MCCs (64.33 5.78 % decrease, p = 0.008, n = 3, Figure 5-3). Following washout, the spiking frequency of the synaptically-isolated MCCs recovered slowly, to 30 8.08 % of control value following 3 hr washout (n = 3).

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121 The difference in decrease by ZD7288 of spik ing frequency of synaptically-coupled and synaptically-isolated MCCs was not signi ficant (p = 0.63, n = 3, Figure 5-4). Knowing that acHCN controls the frequency of the neuronal spiking in individual neurons allows proceeding to determine the role of the channel on systemic level. 5.3.3 Effect of ZD7288 on Systemic Level To determine the role of acHCN on systemic le vel, I blocked the channels by perfusing the preparation with ZD7288 in normal AS and simu ltaneously recorded fr om two symmetrical neurons (two B3s or two P4s) in the left a nd right buccal and pedal ganglia, respectively. ZD7288 significantly decreased the spiking fr equency of B3 neuron (53.33 5.81 % decrease following perfusion with AS containing ZD7288, compared to control, p = 0.01, n = 3, Figure 5-5). Following washout, th e spiking frequency of the B3 neuron recovered slowly, to 41.67 21.48 % of control value following 2 hr washout (n = 3). Similarly, ZD7288 significantly decreased spik ing frequency of the pedal locomotory neuron P4 (94 3.67 % decrease following perf usion with AS containing ZD7288, compared to control, p = 0.0001, n = 4, Figure 5-6). Followi ng washout, the spiking frequency of the P4 neuron recovered slowly, to 23 5.31 % of c ontrol value following 2 hr washout (n = 4). ZD7288 also inhibited the rhythmic activity of unidentified buccal neurons representing the feeding central patte rn generator (Figure 5-7) and t onic activity of unidentified pedal locomotory neurons (not shown). This indicates a potential role of acH CN in triggering and maintenance of feeding and locomotion. 5.4 Discussion Lack of the effects of ZD7288 on different aspe cts of spiking of R15 neuron that does not show detectable expression of acHCN implies th at this inhibitor is specific for the acHCN channels. This finding allowed usi ng ZD7288 to block the channels in A. californica neurons and

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122 thus determine the functional role of acHCN As predicted, ZD7288 decreased the spiking frequency of all recorded neurons expressing the channel, i.e., M CCs, B3s and P4s, implying that acHCN controls the frequency of neuronal spiking. This reduction of spiking frequency is not caused by decreased synaptic input from other neurons because in Hi-Di solution ZD7288 decreased spiking frequency by approximately th e same amount as in the normal saline. The recovery of the spiking frequency upon washout was slow, with only a partial recovery following 3 hours of washout. One of the e xplanations for this is that the volume of the Petri dish containing the preparation is relativel y large, much larger than the volume of the oocyte chamber. Consequently, the inhibitor is washed out slowly. The other explanation is that the recovery of the spiking frequency following washout is due to the synthesis of the new channels. I consider this explanation more pl ausible, because in vertebrates ZD7288 was shown to be irreversible (B oSmith et al., 1993). Since B3 and P4 neurons belong to feeding and locomotory networ ks, respectively, the reduction in the spiking frequency of these ne urons by ZD7288 implies th e role of acHCN in controlling activity of the feeding and locomotory networks. In addition, because spiking of B3 and P4 neurons directly correlate s with the contraction of buccal and pedal muscles, respectively (Church and Lloyd, 1994; Hening et al., 1979), the re sults of the described experiments implicate the role of acHCN in controlling feeding and locomotion in A. californica This role is also supported by inhibition by ZD7288 of the rhythmic activity of different unidentified buccal neurons representing the feeding central pattern ge nerator and tonic activity of unidentified pedal locomotory neurons. A similar role for an HCN channel in controlling spiking frequency in identifiable feeding motoneurons, specifically, B4/B8 and B4CL (Staras et al., 1998), was suggested in L. stagnalis Serotonin increased the firing fr equency of these neurons, and this effect was due to the

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123 activation of a Cs+-sensitive current and decreased th e duration of posti nhibitory rebound, membrane depolarization occurring at the offs et of hyperpolarization (Straub and Benjamin, 2001). Thus, this current was presumably identified as Ih; however, it was not further characterized. Therefore, it may be a different Cs+-sensitive current. Similarly, Bertrand and Cazalets (1998) showed that postinhibitory re bound in the rat locomotory motoneurons was abolished by Cs+ and experimental regulation of the pos tinhibitory rebound m odulated spiking of these neurons. But again, the Cs+-sensitive current was not further characterized. Also, Chaplan et al. (2003) showed a role for HCN channels in the increased spiking of the rat dorsal root ganglion cells following nerve injury and associated behaviors, i.e., pawlicking, but this work was not done on the identifiable cells. Thus, to the best of my knowledge, my work is the first to show the role of an HCN channel on the molecu lar level, the level of individual identifiable neurons and a systemic level. One of the future goals woul d be to determine under what conditions the activity of acHCN changes. For example, the activity of acHCN and the spiking frequency of A. californica neurons are expected to change when the concentr ation of cyclic nucleotides changes. One such change occurs during nerve injury (Siegan et al., 1996). Elevated cGMP may activate acHCN that would in turn depolarize ne urons and lead to the increased fr equency of their spiking, one of the most important manifestations of the LTH. Elevated cAMP is not expected to activate acHCN in the same way as cGMP, because in oo cyte experiments cAMP did not change acHCNmediated current. However, since cAMP is one of the main transcriptional regulators it may decrease transcription of acHCN, thus preventing excessive excitability. In addition to being activated by cGMP, the acHCN channel may produce larger currents due to up-regulation of the synthesis of acHCN protein following nerve injury as in the rat dorsal root ganglia (Chaplan et al., 2003). Alternatively, if acHCN protein is down-regulated follo wing nerve injury, similar to

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124 the acHCN mRNA, this may decrease the acHCN-m ediated currents and reduce the frequency of neuronal spiking. These possibi lities can be tested by perfor ming immunohistochemistry and electrophysiologically recording Ih both under normal conditions and following nerve crush. Also, increased spiking of neurons following nerve injury in A. californica may not be due to activation of HCNs, but other channels, e.g., Na+ channels. Based on the properties of acHCN, it may also pl ay a role in classical conditioning. It was long known that this process in A. californica is mediated by serotonin and cAMP (Kandel, 2001). However, recently it has been shown that another mechanism of classical conditioning in Aplysia is through nitric oxi de (Antonov et al., 2 007). Nitric oxide in Aplysia activates guanylyl cyclase and thus increases production of cGMP (Bodnrov et al., 2005). cGMP, in turn, is expected to activate acHCN, which will increase the excitability of the neurons. HCN channels were also suggested to play an important role in LTP at the neuromuscular junction of the crayfish (Beaumont and Zucker, 2000). In this sy stem, HCN channels were shown to act at the presynaptic level by depolarization of the presynaptic membrane. Because in A. californica HCN channels are expressed predominantly in motone urons and not in the sensory neurons, acHCN may act postsynaptically at the le vel of sensorimotor c onnections or presynap tically at the level of neuromuscular connections.

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125 Figure 5-1. Determination of the sp ecificity of ZD7288 for acHCN. ZD7288 (150 M) did not influence any aspect of spiking of R15 ne uron that does not express acHCN. A) R15 recording in control. B) R15 recordi ng following perfusion with AS containing ZD7288. C) Number of spikes per minut e did not significantly change following perfusion with AS containing ZD7288, compared to control (1.19 0.40 fold change, p = 0.68, n = 3). D) Spikes amplitude did not significantly change following perfusion with AS containing ZD7288, compared to control (1.01 0.01 fold change, p = 0.64, n = 3). E) Maximal hyperpol arization did not si gnificantly change following perfusion with AS containing ZD 7288, compared to control (0.95 0.06 fold change, p = 0.51, n = 3). Spikes per min / Spikes per min in Ctrl 0.0 0.4 0.8 1.2 1.6 2.0 Ctrl ZD7288 Spike ampl / Spike ampl in Ctrl 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288 Max Hyperpol / Max Hyperpol in Ctrl 0.0 0.2 0.4 0.6 0.8 1.0 Ctrl ZD7288C D E 80 60 40 20 0 -50 0 50 -50 0 50 80 60 40 20 0A BVm, mV Vm, mVTime, s Time, s Spikes per min / Spikes per min in Ctrl 0.0 0.4 0.8 1.2 1.6 2.0 Ctrl ZD7288 Spike ampl / Spike ampl in Ctrl 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288 Max Hyperpol / Max Hyperpol in Ctrl 0.0 0.2 0.4 0.6 0.8 1.0 Ctrl ZD7288C D E 80 60 40 20 0 -50 0 50 -50 0 50 80 60 40 20 0A BVm, mV Vm, mVTime, s Time, s

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126 Figure 5-2. Effect of ZD7288 on spiki ng of the metacerebral cells. ZD7288 (150 M) significantly decreased spiking frequency of MCCs (70 5.03 % decrease following perfusion with AS containing ZD7288, compared to control, p = 0.005, n = 3). Following washout, the spiking frequency reco vered very slowly, with a recovery of 26.50 8.50 % of control value following 2 hr washout (n = 3) and 58 % of control value following 3 hr washout (n = 1). A) MCC recording in control. B) MCC recording following perfusion with AS so lution containing ZD7288. C) Graphical representation of the ef fect of ZD7288 on spiking frequency of MCCs. A B -50 0 50 20 0 -50 0 50 20 0Vm, mV Vm, mVTime, sTime, s Spike number / Spike number in Control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288 1hr 2hr 3hr* W a s h o u t -50 0 50 20 0 -50 0 50 20 0Vm, mV Vm, mVTime, sTime, s Spike number / Spike number in Control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288 1hr 2hr 3hr* W a s h o u tC

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127 Figure 5-3. Effect of ZD7288 on spiking of th e synaptically-isolated metacerebral cells. ZD7288 (150 M) significantly decreased spiking frequency of the synapticallyisolated MCCs (64.33 5.78 % decrease following perfusion with Hi-Di solution containing ZD7288, compared to control, p = 0.008, n = 3). Following washout, the spiking frequency recovered slowly, to 30 8.08 % of control value following 3 hr washout (n = 3).. A) MCC recording in control. B) MCC recording following perfusion with Hi-Di solution containing ZD 7288. C) Graphical representation of the effect of ZD7288 on spiking frequency of the synaptically-isolated MCCs. Spike number / Spike number in Control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288 1 hr2 hr 3 hr W a s h o u t -50 0 50 20 0 -50 0 50 20 0A BTime, sTime, sVm, mV Vm, mVC Spike number / Spike number in Control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288 1 hr2 hr 3 hr W a s h o u t -50 0 50 20 0 -50 0 50 20 0 -50 0 50 20 0 -50 0 50 20 0A BTime, sTime, sVm, mV Vm, mVC

PAGE 128

128 \ Figure 5-4. Comparison of the effect of ZD7288 on spiking of synaptically-coupled and synaptically-isolated MCCs. The difference in decrease by ZD7288 (150 M) of spiking frequency of synap tically-coupled and synaptically-isolated MCCs was not significant (p = 0.63, n = 3). Spike number / Spike number in Control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288 1hr 2hr W a s h o u t

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129 Figure 5-5. Effect of ZD7288 on sp iking of B3 neuron. ZD7288 (150 M) significantly decreased spiking frequency of B3 ne uron (53.33 5.81% decrease following perfusion with AS containing ZD7288, compared to control, p = 0.01, n = 3). Following washout, the spiking frequency of the B3 neuron recovered slowly, to 41.67 21.48 % of control value following 2 hr washout (n = 3). A) B3 recording in control. B) B3 recording following pe rfusion with AS containing ZD7288. C) Graphical representation of the effect of ZD7288 on spiking frequency of B3 neuron. -50 0 50 0 5 Spike number / Spike number in Control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288 1hr 2hr W a s h o u t* -50 0 50 0 5Vm, mV Vm, mVTime, sTime, sA B C -50 0 50 0 5 Spike number / Spike number in Control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288 1hr 2hr W a s h o u t* -50 0 50 0 5Vm, mV Vm, mVTime, sTime, sA B C

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130 Figure 5-6. Effect of ZD7288 on spiking of the pedal locomotory neuron P4. ZD7288 (150 M) significantly decreased spiking frequenc y of P4 neuron (94 3.67 % decrease following perfusion with AS containing ZD 7288, compared to control, p = 0.0001, n = 4). Following washout, the spiking frequenc y of the P4 neuron recovered slowly, to 23 5.31 % of control value following 2 hr washout (n = 4). A) P4 recording in control. Upper trace P4 in the left pedal ga nglion; lower trace P4 in the right pedal ganglion. B) P4 recording following pe rfusion with AS containing ZD7288. Upper trace P4 in the left pedal ganglion; lower trace P4 in the right pedal ganglion. C) Graphical representation of the effect of ZD7288 on spiking frequency of P4 neuron. Spike number / Spike number in Control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288* 1hr 2hr W a s h o u t -50 0 50 -50 0 50 40 20 0 40 20 0 -50 0 50 -50 0 50 Vm, mV Vm, mVTime, sTime, s Spike number / Spike number in Control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ctrl ZD7288* 1hr 2hr W a s h o u t -50 0 50 -50 0 50 40 20 0 40 20 0 -50 0 50 -50 0 50 Vm, mV Vm, mVTime, sTime, sA B C

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131 Figure 5-7. Effect of ZD7288 on the rhythmic activity of two unide ntified buccal neurons expressing acHCN transcript. ZD7288 (150 M) inhibits rhythmic activity of the unidentified buccal neurons. A) Control. B) Perfusion with ZD7288. C) 3 hr washout.

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132 CHAPTER 6 SUMMARY AND FUTURE WORK The main objective of my PhD work was to determine the role of acHCN in neuronal excitability. As the first step for achieving this goal, I have cloned a coding region of the channel from the CNS of A. californica The evidence presented in Chapter 2 suggests that Aplysia has only one HCN channel and that the whole coding region was obtained. I did not reach poly-A tail at the end of the transcript because existence of the long 3-UTR, which is almost identical to the coding region of acHCN, made cloning of the 3-UTR very difficult. Completion of the A. californica genome will help to determine the end of the acHCN transcript and to fill the gaps in the current genomic representation of the transcript. The latter will allow determining the number of exons in the acHCN gene and compar ing it with HCN genes from other organisms. The available sequence of acHCN, however, is su fficient to compare this channel with the HCN channels from other organisms. This analysis described in section 2. 3.1, shows that the 5region of the cloned acHCN channel is different from other HCN channels. Due to a singlenucleotide mutation, acHCN has an ATT sequence at the position where HCN channels of other organisms have their first ATG (start c odon). The first ATG codon of acHCN is 28 aa downstream, thus the channel is possibly truncated at its N-term inus. When in evolution the ATG ATT mutation occurred and whether HCN channe ls of other invertebra te phyla are also mutated in this position can be determined wh en genomic sequencing and annotation for species from other invertebrate phyla will be completed. The existence of two potential phosphoryla tion sites and a weak alignment between acHCN and other HCN channels just upstream of the first ATG sequence of acHCN (see Figure 2-2) suggest that translation of acHCN may start from the altern ative start codon, CTG, upstream of the first ATG codon (see secti on 2.3.1). To determine the tran slation start of acHCN, Western blot using antibodies to the acHCN protein has to be performed. This Western blot could also

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133 demonstrate whether a shortened form of acHCN (see section 2.3.3) without the CNBD is synthesized. Having antibodies to the acHCN channel will also allow localizing the protein in the CNS of A. californica and determining how the expression of the protein changes under different conditions, e.g., following nerve injury. Therefor e, we are planning to obtain these antibodies and perform the above listed experiments. Because of the possible N-terminal truncation of acHCN, the question arose of whether the cloned channel is functional. To answer this question, the acHCN chan nel was expressed in X. laevis oocytes. The properties of the channel, namely, its activation by both hyperpolarization and cyclic nucleotides, permeability to pota ssium and sodium ions and inhibition by Cs+ and ZD7288, confirm that a functional form of acHCN was cloned. Finding that acHCN is inhibited by ZD7288 allowed me to remove the acHCN-media ted current and determine how the channel influences neuronal spiking. Model cells for this study, cerebral inte rneurons MCC, buccal motoneurons B3 and pedal locomotory neurons P4 were chosen following localization of the acHCN transcript in th e CNS of the animal. Electrophysiological recording of acHCN in the A. californica neurons demonstrated that the channel controls the frequenc y of neuronal spiking in the targ et neurons and thus may control the associated behaviors, i.e., feeding and loco motion. However, it remains to be determined, under what conditions acHCN is ac tivated or inhibited. One of the possible conditions is nerve injury. We will determine how acHCN-mediated current changes following nerve crush in the neurons of feeding and locomotory networks. Properties of HCN channels from different an imal phyla are summarized in Table 6-1. As most other HCN channels, acHCN is activated by both hyperpolarization and cyclic nucleotides and passes both K+ and Na+. However, unlike the HCN channels of vertebrates, arthropods and echinoderms, the voltage of acHCN activation is not shifted in de polarized direction by cyclic

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134 nucleotides. Another unique featur e of acHCN is that cGMP is more effective than cAMP in activating the channel. In additi on, unlike vertebrates and arthr opods, acHCN is not expressed in sensory neurons.

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135 Table 6-1. Comparison of acH CN and other HCN channels Property Vertebrates Arthropods* Echinoderms** Aplysia Activation by hyperpolarization + + + Permeability to K+ and Na+ + + + + Activation by cNMPs + + + + Shift of V1/2 by cNMPs + (except HCN3) + + More effective cNMP cAMP cAMP cAMP cGMP Localization in CNS SN, MN SN, MN ? MN D. melanogaster A. mellifera P. argus and P. interruptus ** S. purpuratus

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136 APPENDIX A SEQUENCES OF THE HCN CHANNELS USED TO CONSTRUCT PHYLOGENETIC TREE AND THEIR ALIGNMENT >acHCN LGPTSGAGIVAGHGNPSITITLDSDSDSVYS DYLSPEINYKAHDPKVQFLGDDTSLYGTPK EELPMGQECVAGEAGGAASKASSTTSYLKDQI LNFFQPSDNKLAMKLFGNKNALIKEK MRHKRVGNWVIHPCSNFRFYWDLFMLVLLIANLIILPVAISFFNDDLSTHWIVFNCISDT VFFLDIVINFRTGIILNDFADEIILDPKLIAK QYMKTWFFLDLLSSVPMDYIFLMWDAEAD FNQLFHAGRALRMLRLAKLLSLLRLLRLSRLVRYVQQWEEFLAIAGKFMRIFNLICLMF LLGHWNGCLQFLVPLIQDFPKDCWVSIEGLQEAHWAEQYTWALFKALSHMLCIGYGRF PPQNMSDTWLTILSMLSGAT CYALFLAHTTTLIQSFDTSRR LYNEKFKQVEEYMVYRKL PRSLRQRITDYYEHRYQGKMFDEETILSEL NECLKHEVVNHNCRSL VASVPFFTNADPAF VSEVVSKLKFEVYQPGDYIIREGTMGTKMFFI QEGIVDIITSDGEVATSLSDGSYFGEICLL TNARRVASVRAETYACLYSLAVEHFTAV LERYPVMRRTMESVAAERLTKIGKNPSIVSS RADLEEDQKMVNEIVMESTPIPTSASEDEDRDSDESSDGSKQKKKTAFKFDFSTKLHKIS EEKKNKSPKEHSKERDLLEFGETKHHRLSQGPDP >hsHCN1 MEGGGKPNSSSNSRDDGNSVFPAKASATG AGPAAAEKRLGTPPGGGGAGAKEHGNSV CFKVDGGGGGGGGGGGGEEPA GGFEDAEGPRRQYGFMQRQFTSMLQPGVNKFSLRMF GSQKAVEKEQERVKTAGFWIIHPYSDFRFY WDLIMLIMMVGNLVIIPVGITFFTEQTTTP WIIFNVASDTVFLLDLIMNFRTGTVNEDSSE IILDPKVIKMNYLKSWSVVDFISSIPVDYIF LIVEKGMDSEVYKTARALRIVRFTKILSLLR LLRLSRLIRYIHQWEEIFHMTYDLASAVVR IFNLIGMMLLLCHWDGCLQFLVPLLQDFPP DCWVSLNEMVNDSWG KQYSYALFKAMS HMLCIGYGAQAPVSMSDLWITMLSMIVGATCYAMFVGHATALIQSLDSSRRQYQEKYK QVEQYMSFHKLPADMRQKIHDYYEHRYQGKI FDEENILNELNDPLRGEIVNFNCRKLVA TMPLFANADPNFVTAMLSKLRFEVFQPG DYIVREGAVGKKMYFIQHGVAGVITKSSKE MKLTDGSYFGEICLLTKGRRTASVRADTYCRLYSLSVDNFNEVPEEYPMMRRAFETVAI DRLDRIGKKNSILLQKFQKDLNTGVFNNQE NEILKQIVKHDREMVQAIAPINYPQMTTLN SASSTTTPTSRMRTQSPPVYTATSLSHSNLH SPSPSTQTPQPSAILSPCSYTTAVCSPPVQS PLAARTFHYASPTASQLSLMQQQPQQQVQQS QPPQTQPQQPSPQPQTPGSSTPKNEVHK STQALHNTNLTREVRPLSASQPSLPHEVPTLISRPHPTVGESLASIPQPVTAVPGTGLQAG GRSTVPQRVTLFRQMSSGAIPPNRGVPPA PPPPAAALPRESSSVLNTDPDAEKPRFASNL >hsHCN2 MDARGGGGRPGESPGASPTTGPPPPPPPRPP KQQPPPPPPPAPPPGPGPAPPQHPPRAEAL PPEAADEGGPRGRLRSRDSSCGRPGTPGAASTAKGSPNGECGRGEPQCSPAGPEGPARG PKVSFSCRGAASGPAPGPGPAEEAGSEEAGPAGEPRGSQASFMQRQFGALLQPGVNKFS LRMFGSQKAVEREQERVKSAGAWIIHPYSD FRFYWDFTMLLFMVGNLIIIPVGITFFKDE TTAPWIVFNVVSDTFFLMDLVLNFRTGIVIEDNTEIILDPEKIKKKYLRTWFVVDFVSSIPV DYIFLIVEKGIDSEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYDLASA VMRICNLISMMLLLCHWDGCLQFLVPMLQD FPRNCWVSINGMVNHSWSELYSFALFKA MSHMLCIGYGRQAPESMTDIWLTMLSMI VGATCYAMFIGHATALIQSLDSSRRQYQEK YKQVEQYMSFHKLPADFRQKIHDYYEHRYQG KMFDEDSILGELNGPLREEIVNFNCRKL VASMPLFANADPNFVTAMLTKLKFEVFQ PGDYIIREGTIGKKMYFIQHGVVSVLTKGNK EMKLSDGSYFGEICLLTRGRRTASVRADT YCRLYSLSVDNFNEV LEEYPMMRRAFETVA IDRLDRIGKKNSILLHKVQHDLNSGVFNNQE NAIIQEIVKYDREMVQQAELGQRVGLFPP PPPPPQVTSAIATLQQAAAMSFCPQVARPLVGPLALGSPRLVRRPPPGPAPAAASPGPPPP

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137 ASPPGAPASPRAPRTSPYGGLPAAPLAGPAL PARRLSRASRPLSASQPSLPHGAPGPAAST RPASSSTPRLGPTPAARAAAPSPDRRDSASPGAAGGLDPQDSARSRLSSNL >hsHCN3 MEAEQRPAAGASEGATPGLEAVPPVAPPPAT AASGPIPKSGPEPKRRHLGTLLQPTVNKF SLRVFGSHKAVEIEQERVKSAGAWIIHPYSDFRFYWDLIMLLLMVGNLIVLPVGITFFKE ENSPPWIVFNVLSDTFFLLDLV LNFRTGIVVEEGAEILLAPRAIRTRYLRTWFLVDLISSIP VDYIFLVVELEPRLDAEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYD LASAVVRIFNLIGMMLLLCHWDGCLQFLVPM LQDFPPDCWVSINHMVNHSWGRQYSH ALFKAMSHMLCIGYGQQAPVGMPDVWLTML SMIVGATCYAMFIGHATALIQSLDSSRR QYQEKYKQVEQYMSFHKLPADTRQRIHEYYEHRYQGKMFDEESILGELSEPLREEIINFT CRGLVAHMPLFAHADPSFVTAVLTKLRFEVFQPGDLVVREGSVGRKMYFIQHGLLSVL ARGARDTRLTDGSYFGEICLLTRGRRTASVRADTYCRLYSLSVDHFNAVLEEFPMMRR AFETVAMDRLLRIGKKNSILQRKRSEPSPGSSGGIMEQHLVQHDRDMARGVRGRAPSTG AQLSGKPVLWEPLVHAPLQAAAVTSNVAIALTHQRGPLPLSPDSPATLLARSAWRSAGS PASPLVPVRAGPWASTSRLPAPPARTLHASLSRAGRSQVSLLGPPPGGGGRRLGPRGRPL SASQPSLPQRATGDGSPGRKGSGSERLPPSG LLAKPPRTAQPPRPPVPEPATPRGLQLSAN M >hsHCN4 MDKLPPSMRKRLYSLPQQVGAKAWIMDEEEDAEEEGAGGRQDPSRRSIRLRPLPSPSPS AAAGGTESRSSALGAADSEGPARGAGKSST NGDCRRFRGSLASLGSRGGGSGGTGSGSS HGHLHDSAEERRLIAEGDASPGEDRTPPGL AAEPERPGASAQPAASPPPPQQPPQPASAS CEQPSVDTAIKVEGGAAAGDQILPEAEVRL GQAGFMQRQFGAMLQPGVNKFSLRMFGS QKAVEREQERVKSAGFWIIHPYSDFRFY WDLTMLLLMVGNLIIIPVGITFFKDENTTPWI VFNVVSDTFFLIDLVLNFRTGIVVEDNTEIIL DPQRIKMKYLKSWFMVDFISSIPVDYIFLI VETRIDSEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYDLASAVVRIV NLIGMMLLLCHWDGCLQFLVPMLQDFPDDC WVSINNMVNNSWGKQYSYALFKAMSH MLCIGYGRQAPVGMSDVWLTMLSMIVGATCYAMFIGHATALIQSLDSSRRQYQEKYKQ VEQYMSFHKLPPDTRQRIHDYYEHRYQGKMFDEESILGELSEPLREEIINFNCRKLVASM PLFANADPNFVTSMLTKLRFEVFQPGDYIIREGTIGKKMYFIQHGVVSVLTKGNKETKLA DGSYFGEICLLTRGRRTASVRADTYCRLYSL SVDNFNEVLEEYPMMRRAFETVALDRLD RIGKKNSILLHKVQHDLNSGVFNYQENEIIQQI VQHDREMAHCAHRVQAAASATPTPTP VIWTPLIQAPLQAAAATTSVAIALTHHPRLPAAIFRPPPGSGLGNLGAGQTPRHLKRLQSL IPSALGSASPASSPSQVDTPS SSSFHIQQLAGFSAPAGLSP LLPSSSSSPPPGACGSPSAPTPS AGVAATTIAGFGHFHKALGGSLSSSDSPLLTPLQPGARSPQAAQPSPAPPGARGGLGLPE HFLPPPPSSRSPSSSPGQLGQPP GELSLGLATGPLSTPETPPRQPEPPSLVAGASGGASPVG FTPRGGLSPPGHSPGPPRTFPSAPPRASGS HGSLLLPPASSPPPPQVPQ RRGTPPLTPGRLT QDLKLISASQPALPQDGAQTLRRASPHSSGESMAAFPLFPRAGGGSGGSGSSGGLGPPGR PYGAIPGQHVTLPRKTSSGSLPPPLSLFGAR ATSSGGPPLTAGPQREPGARPEPVRSKLPS NL >paHCN MNYRDVSKVHFGGDDVSLYGTPKEELGPGQ LCVGAAGAPPGVEPKPSFLKNQLQALFQ PTDNRLAMKLFGSKKALMKERIRQKAAGHWIIHPCSNFRFYWDLCMLFLLVANLIILPV AISFFNDDLSTRWIAFNCLSDTIFLIDIVVNFRT GIKQQDNSEQVILDPKLIARHYLKTWFL LDLISSVPLDYIFLIFNKDFNESFQILQAGRALRILRLAKLLSLVRLLRLSRLVRYVSQWEE VYVSSFLNMASVFMRIFNLISMMLLIGHWSGC LQFLVPMLQGFPSNSWVAINELQSSHW LEQYSWAFFKAMSHMLCIGYGSFPPQNLTDLWLTM ISMISGATCYALFIGHATNLIQSLD SSRRQYRERLKQVEEYMAYRKLPRELRTRI TEYFEHRYQGKFFDEEMILGELSEKLRED VINFNCRALVASV PFFANADARFVTDVVT KLRYEVYQPGDIIIKEGTIGNKMYFIQEGIV

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138 DIVMSNGEVATSLSDGSYFGEICLLTNARRTAS VRAETYCNLFSLSVEHFNTVLDSYPLM RRTMESVAAERLNKIGKNPSIVSNREDLTNDC KTVNAIVNALASVASTEQCDGGTSSEES MMGHDGSSMKGGGGGGGGRH HHHHHHHHNLLDLGSIGKAL AKGHLPRPKSENNFAL SLDTPSPLNRNRPSFHKSDTFHKALRSNSRAPPPMH >dmHCN MHVKHTQRRVSGPGAFGTFTNDQRASRD NLCPDNQQQQQQRHSHSHQHLVRQSLVSL SNGQPAASAAPDSVSLLRAGDDQQQHPQQP HLQQQQQQQQHLQQQQQRHRSSSSRLST SGISKQNSSDSRSGLRILDSSHSPVSCGTQSVSSTGGQSALYDACHEYSRSLSAAAAEGA ASLLKSHYSDQQLAQTEPDPEPDPERDRDRDRDRDRDRERERRHLTNLNLNLSSEYDYS GSDKQQLVNETYIFKCIANSPSFLRTNKIK EQSKKLRNLSLKTRTAKKKGQIISKSNAVSD NSLHPGDKYLNLYLVEKKHSLQPQVAST SSSINTTHPQASSAPASSSSTCTKAPQARQQQ LLLNGSLKGKGQSQSQGQSRQTLP GHRASVRSESGSGSSHTI PATGKSPPVPHSLAAKISS SASGSKNCNLLSASSNSCHKLNAHAQGSGAGS GSGSGSGSGPPGHSHYAAASPKSSVSS NGHLNKYCLTDLTRRKAEFNRQLSAPTDYT HHSSSNGSQQEGSSEANEGHEPVGESTIT VASAGVSYPHPYSYPYHYAHHA SSATAPANLKASLQLHSFGSHHPCPYPARPTSTSCTN SFNRRHIRRHKGKLGDRLLSGDSEESVRCSYC SVLNVNDNDLRISFENTCTDSLVTAFDD EALLICDQGTEMVHFDDVSLYGTPKEEPMPNI PIVSEKVSANFLKSQLQSWFQPTDNRLA MKLFGSRKALVKERIRQKTSGHWVIHP CSSFRFYWDLCMLLLLVANLIILPVAISFFNDD LSTRWIAFNCLSDTIFLIDIVVNFRTGIMQQ DNAEQVILDPKLIAKHYLRTWFFLDLISSIPL DYIFLIFNQDFSDSFQILHAGRALRILRLAKLLSLVRLLRLSRLVRYVSQWEEVYFLNMA SVFMRIFNLICMMLLIGHWSGCLQFLVPML QGFPSNSWVSINELQESYWLEQYSWALFK AMSHMLCIGYGRFPPQSLTDMWLTMLSMISGATCYALFLGHATNLIQSLDSSRRQYREK VKQVEEYMAYRKLPRDMRQ RITEYFEHRYQGKFFDEELILG ELSEKLREDVINYNCRSL VASVPFFANADSNFVSDVVTK LKYEVFQPGDIIIKEGTIGTKM YFIQEGVVDIVMANGEV ATSLSDGSYFGEICLLTNARRVASVRAETY CNLFSLSVDHFNCVLDQYPLMRKTMETVA AERLNKIGKNPNIMHQKDEQLSNPESNTI TAVVNALAAEADDCKDDDMDLKENLLHGS ESSIAEPVQTIREGLPRPRSGEFRALFEGNTP >lgHCN MPCLQSGTANPSITITLDSDSDSVYSDYLSP EINYKNDVRVQFIG DDTSLYGTPKEELLPP SQETNNAVEIKPSPTSYLKDQILYFFQPSDNK LAMKLFGNKNALLKEKMRHKRVGNWV IHPCSNFRFYWDLVMLVLLIGNLIILPVAI SFFNDDLSTHWIVFNCISDTVFFLDIIINFRTG VILNDFADEIILDPKLIAKHYMKTWFFLDL LSSIPMDYIFLMWDAEADFSQLFHAGRALR MLRLVKLLSLLRLLRLSRLVRYVQQWEEV CFLAIAGKFMRIFNLICLMFLLGHWNGCLQ FLIPMLQDFPKDCWVSIEELQDAHWAEQYTWSL FKALSHMLCIGYGRFPPQNMSDTWL TILSMLSGATCYALFLAHTTTLIQSFDTSRR LYNEKFKQVEEYMIYRKLPRSLRQRITDYY EHRYQGKMFDEETIHRELNECLRQEVINHNCRALVASVPFFTNADPEFVSEVVSKLEFEV YQNGDYIIKEGTIGTKMYFIQEGIVDIITSD NEVATSLSDGSYFGEICLLTNARRVASVRAE TYVNLYSLSVHHFNAVLDRYPVMRRTMESVAAERLTKIGKNPSIVSSRADLEEDQKLVN EIV >CspHCN LDLISSIPLDYIILLFSPRHNF NILPFSFAGRALRIFRLVKLLS LLRLLRLSRLVRYVSQWEE FLTVASKFMRILNLVALMLLLGHWNGCLQWLISLRCNFEHYFDSLISCGMVIRAHWSEQ YTWALFKAMSHMLCIGYGKFPPQSTTDVWLTMISMLTGATCYALFVGHATTLIQSFDTS KRLYREKFKQVEEYMMYRKLPRNLRQRIT DYYEHRYQGKMFDEDSILGELNECLREEIV NYNCRALVASVPFFTHADPNFVSEIITQLKYEV FQPGDYVIKEGTIGTKMYFIQEGIVDIV TKNGEVATSLSDGSYFGEICLLTNAKRVASVRVETYCNLFSLSVEHFNSVLEHYPLMRR TMESVAAERLNKIGKNPNMV >ciHCNa

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139 YIFRYSGGSMSVPVTKFSLRM YGSQKAVEQEQKRQESAGSFV IHPYSNFRFYWDFFTLIL LLISMIIIPVAITFFNDEMRTDSGWIAFNLCLDF WFLSDIIMNFHTGIIVEYGDGDVVLDLP TIRSRYLRSWFVIDLISTLPVDYLLQLTSGS SASASASRAMKLVRFAKIISLLRLLRISRLIR YVHQWEHIVGMQYDLAVAAVRIFNLVCLMLLI GHWNGCLQFLIPMLHNYPADSWIVID KLVGKPWAEQYSWSMFKAMSHMLCIGYGQQPPKNLMDLWMTMLSMVSGAVCFAMFI GHATALIQSMDSSKRQYKEKYMQVKEYMRFRRLPKTLRSKVYEYYENRFQGKMFDEN GILSELSTNLREEVVNFNCRHLVASVPFFS NAESDFVTEVVQKLKYEVFQPKDVIVREGE IGKKMYFIQHGLVEVKNSHRSEPIKQLSDGS YFGEICLLINDRRVASVEAVSYCSTYSLH VNDFNYLLSEHPVMRKTLERVAAARLSSLGK >ciHCNb EPTKMPPKRKVGSKPNESKNDPTPEVPKIFL ATESSSDVPQFSVQVDDHDVEASTNNLTN NMVIASEPTVSYLAMNMDPGLVRRKSQQFEANIKAEEEEHVREALEKERKQVEKLVSS RSVTQGSISSAMHLHPDKSSFRRGVTGSMSIPMNKMSLKLYGSEKAVIEEQERLKKAGS WIIHPYSNFRFIWDSFTLVLLLVNIILIPVIIS FWKDDDSAWLPFKAV SDTWFLTDIIINFRT GIVIDGPDSEVILDPKQIRMMYLKTWFTIDLVSTFPFDLVFTIIVSDGASSMAETGLKALS LLRFAKILALLRLLRLSRFIRYMKQWEEIFNF QYEMALAFARIFNLIMLMLFICHFNGCLQ YMVPMFLDFPEKTWVRDRNLHLSNVTIWERYSWSVFKSTSHMLCIGYGLFPPQGLVDV WVTYVSMCSGAMCFALFIGNATSLIQSMDASKRAYKEKYMQVKEYMQFRKLPSGLRH RISDYYENRFQGKMFDEERILTELNHNLR DEVIQYNCKDLVDQVPFFNEADPSFVAAML GKLNFEVFLNDEVIVKEGTEGKKMYFINRG TVTIKSAQHKIEQSLSDGCYFGEIALLQQN LRRVASVTAETYCYLYSLSVDDFN EVLKEFPRQRAKLQHVAQARL >ciHCNc KWSRKLYGSEKAVQDEHLRVI EAGGFIIHPFSNFRFTWDLL SALLIIANIIIIPMDLAFSGD RREVASMAFKLISDAWFLIDIILNFRTGISVIG TDSTIIELDPAKIRNRYLKGWFAIDFIASF PMDFILTYVVGQAPGKSHKAI SLLRVGKGLSLIRVARIPRLIRGLHQWEEVFNLQYDMA VSLLRLAYLIFIIFLVCHWMGCLQYMVPMYYGFPEDTWVRMRGLDNPNITWWEAYSW SLFKSTSHMLCIGYSEVIPIGLIDLWMTM LSQIVGAILFAVFIGNAINLMEEMDASKNAYK MKLSQITEYLAFRRIPVKLRRKILDYFDIRYTGRLFDEEKILKELSPG >drHCN1 MDEAEDGDEDHKETRRGDILDSSSKMKTVAP GGGAASSRISNNKDFASCGRSTESAALL GHGDASRSGAHSDGEDATGMRCVINGDCRRDDS VCSVLSKLEKQTGGGFPASACHSST SSMDGSVTPAAAAAPPADKKDSRVSFSSAAPA HGPSPSNPAGNSVSFSKSEDGQITAEDG EARDNQTYMQRQFSAMLQPGVNKFSLRMFGSQKAVEKEQERVKSAGNWIIHPYSDFRF YWDFTMLMFMVGNLIIIPVGITFFKEETTTPWIIF NVVSDTFFLMDLVLNFRTGIVYEDNT EIILDPEKIKKKYLKTWFVVDFVSSIPVDYIF LIVEKGIDSEVYKTARALRIVRFTKILSLLR LLRLSRLIRYIHQWEEIFHMT YDLASAVMRIINLIGMMLLLCHWDGCLQFLVPMLQDFPS DCWVSLNKMVNDSWSELYSFALFKAMSHMLCIGYGRQAPESMSDIWLTMLSMIVGAT CYAMFIGHATALIQSLDSSRRQYQEKYKQVEQYMSFHKLPADFRQKIHDYYEHRYQGK MFDEESILEELNEPLREEIVNFNCRKLVASMPLF ANADPNFVTAMLTKLRFEVFQPGDYII REGTIGKKMYFIQHGVVSV LTKGNIGMKLSDGSYFGEIC LLTRGRRTASVRADTYCRLY SLSVDNFNEVLEEYPMMRRAFETVAIDR LDRIGKKNSILMHKV QHDLNSGVFNNRENE MIQEIVKYDREMVKLVKQGDMQRPRAMSM TPSTHGSMFGPSSQPSTSSAIATLQQAVA MSFCPQMGNPLMGSGSVQSPRMVRRFQVVQT QTPPGSQYSTLARASALASTPVHSPLA TTARTFQYGSSPPGAPSGSQLSLVQQLVPSPTHRPAVHRSTLTQDARALSASQPSLRPDM IQPVAPSPPQSTRVSSTSIGPPQNLPGPTGL RGSIPPRMALTHQMSVGAFPPASLPQMRPSL DSGLPKKDSISSLPETEQHHIRSRLSSNL >drHCN4

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140 MAWFYWDLTMLLLMVGNLIIIPVGITFFKDE HTPPWIVFNVVSDTFFLMDLVLNFRTGIV KEDNAEIILDPQQIKIKYLRSWFVVDFISSIP VDYIFLIVETRIDSDFYKTARALRIVRFTKIL SLLRLLRLSRLIRYIHQWEEIFHMTYDLAS AMVRIVNLIGMMLLLCHWDGCLQFLVPML QDFPADCWVAKNKMVNDTWGQQYSYALF KAMSHMLCIGYGMYPPVGMTDVWLTILS MIVGATCYAMFVGHATALIQSLDSSRRQYQ EKYKQVEQYMSFHKLPADMRQRIHDYY EHRYQGKMFDEESILGELNEP LREEIINFNCRKLVASMPLFANADPNFVTSMLTKLRFEV FQPGDYIIREGTIGKKMYFIQHGVVSVLTK GNKETKLSDGSYFGEICLLTRGRRTASVRA DTYCRLYSLSVDNFNEVLEEYPMMRRAFETVALDRLDRIGKKNSILQHKVQHDLNSGV LNYQESEIIQQIVQHDRDMAHCANLLQSPSP PAPPSPTPVIWAPLIQAPLQAAAATTSVAI ALTHHPHLPATLFRPPVSLLGSRNEPPSRLKR FQTVAPRTGSTTDSPSTSPSKLHSGVDTP LLASLHTQHSSVTITPSATTNQPVSFRSFSSPSA SPTLSTAQLHPQPRQKQPSTPPLSARLQ AAGAHPPGILTTASSNTSALSAGLSTHSPTHS YPPPKHPQTGSLQFASGGGKSGLTLFHSP PPGSPISIHSQAPESSTSSQTQPSFSSYLALERSA LASIAQYGSANASPSYTPLALSPTVQSP VTGRTFQYSDPSSAAGSHTSLLLPQSSCPGQL PGHHSSGTDSPLGRFYEDLNILSSSHPSL GVEGAGQSSPGYLSPYLSPTLTPKPCSSVSGH MSSPVQTSPGPRGQALSVGTSPALSLPRT SDGDLEPLRSKLPSNL >amHCN MNYKGSGKVHFGVDDVSLYGTPKEEPGP GLPGQEVKQSFLKNQLQALFQPTDNKLAM KLFGSKKALMKERIRQKAAGHWVIHPCS SFRFYWDLCMLLLLVANLIILPVAISFFNDDL STRWIAFNCLSDTIFLIDIVVNFRTGIMQQ DNAEQVILDPKLIAKHYLRTWFFLDLISSIPL DYIFLIFNQFQDFSESFQILHAGRALRILRLAKLLSLVRLLRLSRLVRYVSQWEEVYFLNM ASVFMRIFNLICMMLLIGHWSGCLQFLVPMLQGFPSNSWVAINELQDSFWLEQYSWALF KAMSHMLCIGYGRFPPQSLTDMWLTMLSMISGATCYALFLGHATNLIQSLDSSRRQYRE KVKQVEEYMAYRKLPREMRQRITEYFEHR YQGKFFDEELILGELSEKLREDVINYNCRS LVASVPFFANADSNFVSDVVT KLRYEVFQPGDIIIKEGTIGS KMYFIQEGIVDIVMANGEV ATSLSDGSYFGEICLLTNARRVASVRAETY CNLFSLSVDHFNAVLDQYPLMRRTMESVA AERLNKIGKNPNLVAHREEDLGSESKTI NAVVNALAEQAAHASASEESVHSMELRTLPC LLPRPKSENNFASQELSREGRRIFHKSDTFHKDSYQ >spHCN1 MDNKETNGELEQSDEADPSGQNLDDGETDS KQEENLINVSPPKTPPGPPPPLKNGGRGQ KPPKIPICHQNGKLPKEVEWTEDRGEDRKDSLTLQSKLDHGAYTDEKQDLLTYLDRHGI NSPVKLTPDETGGSSALDILGIIEERDTGALGSDPSSTMQAMAKPVGFLQRQLWTVLQPS DNRLSMKLFGSKKGLQKEKYRLRKAGVLIIHPCSHFRFYWDLLMLCLIMANVILLPVVI TFFHNKDMSTGWLIFNCFSDTFFILDLICNFRTGIMNPKSAEQVILNPRQIAYHYLRSWFII DLVSSIPMDYIFLLAGGQNRHFLEVSRAL KILRFAKLLSLLRLLR LSRLMRFVSQWEQAF NVANAVIRICNLVCMMLLIGHWNGCLQY LVPMLQEYPDQSWVAINGLEHAHWWEQY TWALFKALSHMLCIGYGKFPPQSITDVWLTI VSMVSGATCFALFIGHATNLIQSMDSSSR QYREKLKQVEEYMQYRKLPSHLRNKILDYYEYRYRGKMFDERHIFREVSESIRQDVAN YNCRDLVASVPFFVGADSNFVTRVVTLLEFEVFQPADYVIQEGTFGDRMFFIQQGIVDII MSDGVIATSLSDGSYFGEICLLTRERRVASVKCETYCTLFSLSVQHFNQVLDEFPAMRKT MEEIAVRRLTRIGKESSKLKSRLESPTIRDTAPLFPIPPDTPSFVTDIEKNRFFGDDTDDVHI RTRVDVERGSHENVIAIMDGSLSDL RMENEIQARKSSSGKRRKFQQQTTEL >spHCN2 MSTAMANKVMPDTQGEFVKVKRPDRLSK ISALRKDFDVKKAAVLKEKPKERRASLPSA MTDKSPGHRDSLVVDLGGAGRRNSMPTKESL FLASGSDFVKKIYRSEKALKDEQERQQ NIRRFVIHPFSNFRWYWDLF MVFLLLITLVLLPVNVTFFSDDITMYWIIINCISDTLFMLDI TLNFFTGVIENAEDTVTLDRTKIIFSSLRGW FFLDLISSFPFDYIYLFLGQVDFTSHTALAL RILRLTKVLSLLRLLRLSRLLRYIHRPEE LLNVETAVIRIVHLVFVMLLLMHWNGCIQFLV

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141 PFFQTFPPDCWVVINGLENAGK LEQYSWSFFKAICHMISIGF GRFPPMNVTEMWMTTFSI MLGATFYALFIGTMSTLLLAVDASGRLYNERL NQVKEYLRYRKVPM NTQRRVLSYYEH RYQRKYFNEKTILGEQSHPIRREILQHHFNN FITKVNFLNEADPDFAYDVIEKLSFEVFLE GDVIIKAGSLGGAMYFIEHGTVEVLVDDRIVNRLSDGDHFGEISLLIDERRVASIIAATTC DVFCLSREDFHKVLKDYPEMGARMAEIAQER LNTIDSSLDTVDEEAGDEEEATANNNN NHPAKNDKKVKKNDLATPQN KDHIQKINDWLKENFAQRI > Nematostella vectensis HCNa DNKLALKLFGNKAAVMLEQRRQSQQGKGVIH PFSTFRWYWDILLIIFIFMHVLLLPVSIA FLSDDLSIHWLILNGVSDVFFVVDIFLNFRT GIVDPNNQEEVILDKKVISMMYLRGWFIID LASSLPFDYAYFIASSTTEEQTLLRASRALR ILKLAKLLSLLRLLRVSRLVRYMTRFEELL NIAKGQLRIMKLICCMLVLSH WNGCIQFFVPYLQEFPDDSWV STSNLKTASPGEQYSWS LFRALSHMLCIGYGHYPPQNLTDLWLTVCSMT AGATFYAVFIGIMSSLIMSIDSSGRLFN EKLNQVEEYMRYRKLPLRIRLQVQDFYEHRF HHKLFDEDAILTELSKNLRETILVHNIKP LLTTVPFFSGASISFITDIVTKLKFEVFLNGD YICRSGHRGDKMYFIQKGIVDILTREGALA TSLGDGSHFGEICLLTKEARRVASVRAATTC DVYSLSATHFHEVLQDYPEMKSVLEEVA KERLSRLGLKPKL > Nematostella vectensis HCNb IEMRLKSWLKPMDNKMNMKVFGSKRAMKDEQLRYSRAGWVIHPTSIFRLYWDMWVL LLLLINLFALPVLISFFADNVSARWIAFNAVSDT AFLLDILLNFRTGVL VHGSPNKFILDPK IIAKRYLKTWFFIDLISSLPIDYIVEAATQSST KSLIGATRTLKLLRFA KLLGLLKLLRLSRL VRYVQQYEEILNVTRSVIRFINLVSIILLI AHWNGCLQFLVPYLQDFPETSWVSIHNLMDN DWWEQYCWSLFKAMSHMLCIGYGRYPPQNI AEVWVTTFSMLTGATFYAMFIAYCINFI QQLDSPGRNYREKIQQIEEYMSYRRLPVELRDRMTKYYDHRYQGRMFDEEKILHEISKP LREQIINYNCRDLVQSVPFFTEAEPDFVSAIIT RLSFEVYLEGDIIVREGELGTEMYFLREG VVSVTVGGKHANELCDGAYFGEICLLTNARRTASVTAKTVCDVFILNAEHFRDVVDEFP TMRILMEIVAEDRLNKMGK

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142 Figure A-1. Alignment of HCN proteins used for construction of the phylogenetic tree. This alignment was obtained using ClustalX fo llowed by the removal of all gaps in GeneDoc. Abbreviations: hs Homo sapiens (human), ac Aplysia californica dr Danio rerio (zebrafish), sp Strongylocentrotus purpuratus (sea urchin), dm Drosophila melanogaster pa Panulirus argus (lobster), am Apis mellifera (honey bee), lg Lottia gigantea (limpet), Csp Capitella sp (polychaete worm), ci Ciona intestinalis (sea squirt).

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143 APPENDIX B SEQUENCES OF THE CNBD-CONTAINING POTASSIUM AND CATION CHANNELS USED TO CONSTRUCT PHYLOGENETIC TREE AND THEIR ALIGNMENT >hsHCN1 MEGGGKPNSSSNSRDDGNSVFPAKASATG AGPAAAEKRLGTPPGGGGAGAKEHGNSV CFKVDGGGGGGGGGGGGEEPA GGFEDAEGPRRQYGFMQRQFTSMLQPGVNKFSLRMF GSQKAVEKEQERVKTAGFWIIHPYSDFRFY WDLIMLIMMVGNLVIIPVGITFFTEQTTTP WIIFNVASDTVFLLDLIMNFRTGTVNEDSSE IILDPKVIKMNYLKSWSVVDFISSIPVDYIF LIVEKGMDSEVYKTARALRIVRFTKILSLLR LLRLSRLIRYIHQWEEIFHMTYDLASAVVR IFNLIGMMLLLCHWDGCLQFLVPLLQDFPP DCWVSLNEMVNDSWG KQYSYALFKAMS HMLCIGYGAQAPVSMSDLWITMLSMIVGATCYAMFVGHATALIQSLDSSRRQYQEKYK QVEQYMSFHKLPADMRQKIHDYYEHRYQGKI FDEENILNELNDPLRGEIVNFNCRKLVA TMPLFANADPNFVTAMLSKLRFEVFQPG DYIVREGAVGKKMYFIQHGVAGVITKSSKE MKLTDGSYFGEICLLTKGRRTASVRADTYCRLYSLSVDNFNEVPEEYPMMRRAFETVAI DRLDRIGKKNSILLQKFQKDLNTGVFNNQE NEILKQIVKHDREMVQAIAPINYPQMTTLN SASSTTTPTSRMRTQSPPVYTATSLSHSNLH SPSPSTQTPQPSAILSPCSYTTAVCSPPVQS PLAARTFHYASPTASQLSLMQQQPQQQVQQS QPPQTQPQQPSPQPQTPGSSTPKNEVHK STQALHNTNLTREVRPLSASQPSLPHEVPTLISRPHPTVGESLASIPQPVTAVPGTGLQAG GRSTVPQRVTLFRQMSSGAIPPNRGVPPA PPPPAAALPRESSSVLNTDPDAEKPRFASNL >hsHCN2 MDARGGGGRPGESPGASPTTGPPPPPPPRPP KQQPPPPPPPAPPPGPGPAPPQHPPRAEAL PPEAADEGGPRGRLRSRDSSCGRPGTPGAASTAKGSPNGECGRGEPQCSPAGPEGPARG PKVSFSCRGAASGPAPGPGPAEEAGSEEAGPAGEPRGSQASFMQRQFGALLQPGVNKFS LRMFGSQKAVEREQERVKSAGAWIIHPYSD FRFYWDFTMLLFMVGNLIIIPVGITFFKDE TTAPWIVFNVVSDTFFLMDLVLNFRTGIVIEDNTEIILDPEKIKKKYLRTWFVVDFVSSIPV DYIFLIVEKGIDSEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYDLASA VMRICNLISMMLLLCHWDGCLQFLVPMLQD FPRNCWVSINGMVNHSWSELYSFALFKA MSHMLCIGYGRQAPESMTDIWLTMLSMI VGATCYAMFIGHATALIQSLDSSRRQYQEK YKQVEQYMSFHKLPADFRQKIHDYYEHRYQG KMFDEDSILGELNGPLREEIVNFNCRKL VASMPLFANADPNFVTAMLTKLKFEVFQ PGDYIIREGTIGKKMYFIQHGVVSVLTKGNK EMKLSDGSYFGEICLLTRGRRTASVRADT YCRLYSLSVDNFNEV LEEYPMMRRAFETVA IDRLDRIGKKNSILLHKVQHDLNSGVFNNQE NAIIQEIVKYDREMVQQAELGQRVGLFPP PPPPPQVTSAIATLQQAAAMSFCPQVARPLVGPLALGSPRLVRRPPPGPAPAAASPGPPPP ASPPGAPASPRAPRTSPYGGLPAAPLAGPAL PARRLSRASRPLSASQPSLPHGAPGPAAST RPASSSTPRLGPTPAARAAAPSPDRRDSASPGAAGGLDPQDSARSRLSSNL >hsHCN3 MEAEQRPAAGASEGATPGLEAVPPVAPPPAT AASGPIPKSGPEPKRRHLGTLLQPTVNKF SLRVFGSHKAVEIEQERVKSAGAWIIHPYSDFRFYWDLIMLLLMVGNLIVLPVGITFFKE ENSPPWIVFNVLSDTFFLLDLV LNFRTGIVVEEGAEILLAPRAIRTRYLRTWFLVDLISSIP VDYIFLVVELEPRLDAEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYD LASAVVRIFNLIGMMLLLCHWDGCLQFLVPM LQDFPPDCWVSINHMVNHSWGRQYSH ALFKAMSHMLCIGYGQQAPVGMPDVWLTML SMIVGATCYAMFIGHATALIQSLDSSRR QYQEKYKQVEQYMSFHKLPADTRQRIHEYYEHRYQGKMFDEESILGELSEPLREEIINFT CRGLVAHMPLFAHADPSFVTAVLTKLRFEVFQPGDLVVREGSVGRKMYFIQHGLLSVL ARGARDTRLTDGSYFGEICLLTRGRRTASVRADTYCRLYSLSVDHFNAVLEEFPMMRR AFETVAMDRLLRIGKKNSILQRKRSEPSPGSSGGIMEQHLVQHDRDMARGVRGRAPSTG

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144 AQLSGKPVLWEPLVHAPLQAAAVTSNVAIALTHQRGPLPLSPDSPATLLARSAWRSAGS PASPLVPVRAGPWASTSRLPAPPARTLHASLSRAGRSQVSLLGPPPGGGGRRLGPRGRPL SASQPSLPQRATGDGSPGRKGSGSERLPPSG LLAKPPRTAQPPRPPVPEPATPRGLQLSAN M >hsHCN4 MDKLPPSMRKRLYSLPQQVGAKAWIMDEEEDAEEEGAGGRQDPSRRSIRLRPLPSPSPS AAAGGTESRSSALGAADSEGPARGAGKSST NGDCRRFRGSLASLGSRGGGSGGTGSGSS HGHLHDSAEERRLIAEGDASPGEDRTPPGL AAEPERPGASAQPAASPPPPQQPPQPASAS CEQPSVDTAIKVEGGAAAGDQILPEAEVRL GQAGFMQRQFGAMLQPGVNKFSLRMFGS QKAVEREQERVKSAGFWIIHPYSDFRFY WDLTMLLLMVGNLIIIPVGITFFKDENTTPWI VFNVVSDTFFLIDLVLNFRTGIVVEDNTEIIL DPQRIKMKYLKSWFMVDFISSIPVDYIFLI VETRIDSEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYDLASAVVRIV NLIGMMLLLCHWDGCLQFLVPMLQDFPDDC WVSINNMVNNSWGKQYSYALFKAMSH MLCIGYGRQAPVGMSDVWLTMLSMIVGATCYAMFIGHATALIQSLDSSRRQYQEKYKQ VEQYMSFHKLPPDTRQRIHDYYEHRYQGKMFDEESILGELSEPLREEIINFNCRKLVASM PLFANADPNFVTSMLTKLRFEVFQPGDYIIREGTIGKKMYFIQHGVVSVLTKGNKETKLA DGSYFGEICLLTRGRRTASVRADTYCRLYSL SVDNFNEVLEEYPMMRRAFETVALDRLD RIGKKNSILLHKVQHDLNSGVFNYQENEIIQQI VQHDREMAHCAHRVQAAASATPTPTP VIWTPLIQAPLQAAAATTSVAIALTHHPRLPAAIFRPPPGSGLGNLGAGQTPRHLKRLQSL IPSALGSASPASSPSQVDTPS SSSFHIQQLAGFSAPAGLSP LLPSSSSSPPPGACGSPSAPTPS AGVAATTIAGFGHFHKALGGSLSSSDSPLLTPLQPGARSPQAAQPSPAPPGARGGLGLPE HFLPPPPSSRSPSSSPGQLGQPP GELSLGLATGPLSTPETPPRQPEPPSLVAGASGGASPVG FTPRGGLSPPGHSPGPPRTFPSAPPRASGS HGSLLLPPASSPPPPQVPQ RRGTPPLTPGRLT QDLKLISASQPALPQDGAQTLRRASPHSSGESMAAFPLFPRAGGGSGGSGSSGGLGPPGR PYGAIPGQHVTLPRKTSSGSLPPPLSLFGAR ATSSGGPPLTAGPQREPGARPEPVRSKLPS NL >dmHCN MHVKHTQRRVSGPGAFGTFTNDQRASRD NLCPDNQQQQQQRHSHSHQHLVRQSLVSL SNGQPAASAAPDSVSLLRAGDDQQQHPQQP HLQQQQQQQQHLQQQQQRHRSSSSRLST SGISKQNSSDSRSGLRILDSSHSPVSCGTQSVSSTGGQSALYDACHEYSRSLSAAAAEGA ASLLKSHYSDQQLAQTEPDPEPDPERDRDRDRDRDRDRERERRHLTNLNLNLSSEYDYS GSDKQQLVNETYIFKCIANSPSFLRTNKIK EQSKKLRNLSLKTRTAKKKGQIISKSNAVSD NSLHPGDKYLNLYLVEKKHSLQPQVAST SSSINTTHPQASSAPASSSSTCTKAPQARQQQ LLLNGSLKGKGQSQSQGQSRQTLP GHRASVRSESGSGSSHTI PATGKSPPVPHSLAAKISS SASGSKNCNLLSASSNSCHKLNAHAQGSGAGS GSGSGSGSGPPGHSHYAAASPKSSVSS NGHLNKYCLTDLTRRKAEFNRQLSAPTDYT HHSSSNGSQQEGSSEANEGHEPVGESTIT VASAGVSYPHPYSYPYHYAHHA SSATAPANLKASLQLHSFGSHHPCPYPARPTSTSCTN SFNRRHIRRHKGKLGDRLLSGDSEESVRCSYC SVLNVNDNDLRISFENTCTDSLVTAFDD EALLICDQGTEMVHFDDVSLYGTPKEEPMPNI PIVSEKVSANFLKSQLQSWFQPTDNRLA MKLFGSRKALVKERIRQKTSGHWVIHP CSSFRFYWDLCMLLLLVANLIILPVAISFFNDD LSTRWIAFNCLSDTIFLIDIVVNFRTGIMQQ DNAEQVILDPKLIAKHYLRTWFFLDLISSIPL DYIFLIFNQDFSDSFQILHAGRALRILRLAKLLSLVRLLRLSRLVRYVSQWEEVYFLNMA SVFMRIFNLICMMLLIGHWSGCLQFLVPML QGFPSNSWVSINELQESYWLEQYSWALFK AMSHMLCIGYGRFPPQSLTDMWLTMLSMISGATCYALFLGHATNLIQSLDSSRRQYREK VKQVEEYMAYRKLPRDMRQ RITEYFEHRYQGKFFDEELILG ELSEKLREDVINYNCRSL VASVPFFANADSNFVSDVVTK LKYEVFQPGDIIIKEGTIGTKM YFIQEGVVDIVMANGEV

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145 ATSLSDGSYFGEICLLTNARRVASVRAETY CNLFSLSVDHFNCVLDQYPLMRKTMETVA AERLNKIGKNPNIMHQKDEQLSNPESNTI TAVVNALAAEADDCKDDDMDLKENLLHGS ESSIAEPVQTIREGLPRPRSGEFRALFEGNTP >paHCN MNYRDVSKVHFGGDDVSLYGTPKEELGPGQ LCVGAAGAPPGVEPKPSFLKNQLQALFQ PTDNRLAMKLFGSKKALMKERIRQKAAGHWIIHPCSNFRFYWDLCMLFLLVANLIILPV AISFFNDDLSTRWIAFNCLSDTIFLIDIVVNFRT GIKQQDNSEQVILDPKLIARHYLKTWFL LDLISSVPLDYIFLIFNKDFNESFQILQAGRALRILRLAKLLSLVRLLRLSRLVRYVSQWEE VYVSSFLNMASVFMRIFNLISMMLLIGHWSGC LQFLVPMLQGFPSNSWVAINELQSSHW LEQYSWAFFKAMSHMLCIGYGSFPPQNLTDLWLTM ISMISGATCYALFIGHATNLIQSLD SSRRQYRERLKQVEEYMAYRKLPRELRTRI TEYFEHRYQGKFFDEEMILGELSEKLRED VINFNCRALVASV PFFANADARFVTDVVT KLRYEVYQPGDIIIKEGTIGNKMYFIQEGIV DIVMSNGEVATSLSDGSYFGEICLLTNARRTAS VRAETYCNLFSLSVEHFNTVLDSYPLM RRTMESVAAERLNKIGKNPSIVSNREDLTNDC KTVNAIVNALASVASTEQCDGGTSSEES MMGHDGSSMKGGGGGGGGRH HHHHHHHHNLLDLGSIGKAL AKGHLPRPKSENNFAL SLDTPSPLNRNRPSFHKSDTFHKALRSNSRAPPPMH >spHCN1 MDNKETNGELEQSDEADPSGQNLDDGETDS KQEENLINVSPPKTPPGPPPPLKNGGRGQ KPPKIPICHQNGKLPKEVEWTEDRGEDRKDSLTLQSKLDHGAYTDEKQDLLTYLDRHGI NSPVKLTPDETGGSSALDILGIIEERDTGALGSDPSSTMQAMAKPVGFLQRQLWTVLQPS DNRLSMKLFGSKKGLQKEKYRLRKAGVLIIHPCSHFRFYWDLLMLCLIMANVILLPVVI TFFHNKDMSTGWLIFNCFSDTFFILDLICNFRTGIMNPKSAEQVILNPRQIAYHYLRSWFII DLVSSIPMDYIFLLAGGQNRHFLEVSRAL KILRFAKLLSLLRLLR LSRLMRFVSQWEQAF NVANAVIRICNLVCMMLLIGHWNGCLQY LVPMLQEYPDQSWVAINGLEHAHWWEQY TWALFKALSHMLCIGYGKFPPQSITDVWLTI VSMVSGATCFALFIGHATNLIQSMDSSSR QYREKLKQVEEYMQYRKLPSHLRNKILDYYEYRYRGKMFDERHIFREVSESIRQDVAN YNCRDLVASVPFFVGADSNFVTRVVTLLEFEVFQPADYVIQEGTFGDRMFFIQQGIVDII MSDGVIATSLSDGSYFGEICLLTRERRVASVKCETYCTLFSLSVQHFNQVLDEFPAMRKT MEEIAVRRLTRIGKESSKLKSRLESPTIRDTAPLFPIPPDTPSFVTDIEKNRFFGDDTDDVHI RTRVDVERGSHENVIAIMDGSLSDL RMENEIQARKSSSGKRRKFQQQTTEL >spHCN2 MSTAMANKVMPDTQGEFVKVKRPDRLSK ISALRKDFDVKKAAVLKEKPKERRASLPSA MTDKSPGHRDSLVVDLGGAGRRNSMPTKESL FLASGSDFVKKIYRSEKALKDEQERQQ NIRRFVIHPFSNFRWYWDLF MVFLLLITLVLLPVNVTFFSDDITMYWIIINCISDTLFMLDI TLNFFTGVIENAEDTVTLDRTKIIFSSLRGW FFLDLISSFPFDYIYLFLGQVDFTSHTALAL RILRLTKVLSLLRLLRLSRLLRYIHRPEE LLNVETAVIRIVHLVFVMLLLMHWNGCIQFLV PFFQTFPPDCWVVINGLENAGK LEQYSWSFFKAICHMISIGF GRFPPMNVTEMWMTTFSI MLGATFYALFIGTMSTLLLAVDASGRLYNERL NQVKEYLRYRKVPM NTQRRVLSYYEH RYQRKYFNEKTILGEQSHPIRREILQHHFNN FITKVNFLNEADPDFAYDVIEKLSFEVFLE GDVIIKAGSLGGAMYFIEHGTVEVLVDDRIVNRLSDGDHFGEISLLIDERRVASIIAATTC DVFCLSREDFHKVLKDYPEMGARMAEIAQER LNTIDSSLDTVDEEAGDEEEATANNNN NHPAKNDKKVKKNDLATPQN KDHIQKINDWLKENFAQRI >acHCN LGPTSGAGIVAGHGNPSITITLDSDSDSVYS DYLSPEINYKAHDPKVQFLGDDTSLYGTPK EELPMGQECVAGEAGGAASKASSTTSYLKDQI LNFFQPSDNKLAMKLFGNKNALIKEK MRHKRVGNWVIHPCSNFRFYWDLFMLVLLIANLIILPVAISFFNDDLSTHWIVFNCISDT

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146 VFFLDIVINFRTGIILNDFADEIILDPKLIAK QYMKTWFFLDLLSSVPMDYIFLMWDAEAD FNQLFHAGRALRMLRLAKLLSLLRLLRLSRLVRYVQQWEEFLAIAGKFMRIFNLICLMF LLGHWNGCLQFLVPLIQDFPKDCWVSIEGLQEAHWAEQYTWALFKALSHMLCIGYGRF PPQNMSDTWLTILSMLSGAT CYALFLAHTTTLIQSFDTSRR LYNEKFKQVEEYMVYRKL PRSLRQRITDYYEHRYQGKMFDEETILSEL NECLKHEVVNHNCRSL VASVPFFTNADPAF VSEVVSKLKFEVYQPGDYIIREGTMGTKMFFI QEGIVDIITSDGEVATSLSDGSYFGEICLL TNARRVASVRAETYACLYSLAVEHFTAV LERYPVMRRTMESVAAERLTKIGKNPSIVSS RADLEEDQKMVNEIVMESTPIPTSASEDEDRDSDESSDGSKQKKKTAFKFDFSTKLHKIS EEKKNKSPKEHSKERDLLEFGETKHHRLSQGPDP >paK+ MIPGLQKIRFRKEEQEKMEDQEIELDALNK VDRQLEMLENSSVWKSKPLRIIQKITIFITR LKHSSTTYRFKLLKKSIFLLIRDKASSFQYYLNNYMLFQKPTRWIQIKYEAHASNPLWW RFWCFLKSDETVLLPADKFLFVWDLLMMLVTIANIFYVPLQLSFNLNEDDMGSIFTLFST LPSCIFLIDLILTFFKGYYDRGILQRNKAK IFRHYIKGDFLLDLAIVLPFILSWMGYSAANY LMLIRMTRVRRTMIVIEEISNFKEKSAII YQLFCLIYSLLLISHFC ACLFHYFALYEVDQGY THTWLHQQNIFDEDLYTKYFNSLYWITITS MTVGYGDIVPVTTPEKILVTIITFLVTGVFG YALGMIQSIFYEMAEQTNINNSRLRLVSNHI KQRGLNTQLQFRVRKYIEYYLQFKQDEEL DLDELMGQLNPKLKQEVQIAMYYRYLKHSKLFGTNFSDDLIKKLCFCIHERTFAPEEVII RKDELPNQLYIVLAGQVKSLILEKSIK RYQQGNLLCEREFFYQDFMQYNIVASSFVQVA YLNLSEFQSIIQNHRSSFEQYRYAIDNTVFG QNTNLIICEACQSHHQFKNCPLVFFKKNTY KVLAACHSNHVQSRQTFARKKSKYKQQ RTGVLNGAYDHIIKMRTQLGVQIDQAFLNKI GYPGYEQQQQSDEEEDKYQSPQARRQSFSQFNDHQHSLSQNTLPQDEHYNDFDIDKVE EYEFYYPHDNITKISKIVNKQQLLQRLLDKVSNKKNLFAGLVFRQVVQNII >teK+ MTILKIPIFAPDSKFIRLWEILIVFITVYNAFLI PFRISFEKRFYGIWILLDLIGDVILIADIFLR FHLGYFEHGEYVEDKKKIAQRYFYKLLRRHLI ASFPGDLIARILLPNSLFIIGLCRCPRLLR LPQFYRIFNRWETNINIEPTLIRMCKLIIFIA LITHWVACGWFLIGSWESNFGESWLINKSL KSVGTRTQYINCLYWAITTLTTVGYGDITP TTEIEIIFTLMVMFLGISMYAYTIGNVSSLIS NLDAAQARYREKLHQIKTYMRENKISPKL QKKIRDYYQYKWIENRDIRDYYIVEELPHP LKTKLALQLHKEVIEKVPIFQGSTSHFVEEI VIALKPEIVPPNEYIIREGNLGNEMYFIKRG LVQVFSEKTGSIYRTMEAGTFFGEISLVYEKRRTASIITLTYCELFILYKNDFKKVLEHYP DFAAHVKKIAKERYKLENKE >rpK+ MRDETPAYLRARHYFYEILESQAQTSRMGVI VNRFIVFFIVLSVGITVMESVPAMREDYG RLFQALELLCLVMFSIEYYIRIWIAPEHLPYHHMSPLRAAWAYMISPQGIVDCISVMPLWI ALFGPDDLRVLIILRMLRLLKFARYSSGMRS LLEVLESERRALMACLVILACATLVSATA MHIAEGHVQPEKFGTIPDAMWWAIVTLSTI GYGDVVPATGIGRMVASATIICGLIMIALP VGIVANAFSEVIHRRDFIVNWS MVARVPLFSHLTAGDIAHIMQLLQARQIERGEVVFRRG EPATAMYFIAEGDVEIELGPEDKGRRIRLGT GHFFGEIAVLKRVERSATVKAVSRTRLLV LDGADLRALIAREPSIARKINQIVEG RTGRNLNLEIADLEGQADVSVEENA >bjK+ MSPPKRSAARAAAAAPTWRRP VAPTVPRRPRRLRRSKKPWR AREAVGLVPRGRGLRDP NLRDRLYELLEHDPLAYSVGS RFIQLIIGVIVLDVVAMVLASVPDLDAQFGALFSAIKVF AVIVFALEYAARLWTVAGHTQRKASALSDRL SYAFSALGIIDLMAFLPAAIVLATGRHA TLAALGVLPFFKLIRYSPAMRSLLAAVHAERRA LIGCIVILIGVVLTFASLLYAIERDVQP DKLGTIPQAMWWAIVTLGTVGYGDVVPVTTLG KFVSVFAIISGFAMIALPVAIISTAFAE

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147 EVKRRDFVVTWGMLARVPLFSHLSASEIADIMRLLRARTIEQGEILVRRGDAATSMYFIT AGEVEIALPTQNVRLTDGTFFGEIALLHKTKRSGTVTATRKTRLLVLDAQDFHALIERMP TLAAHVHKTAKARLEETGDL AAAELAQAEREGTDR >mmK+ MINWLYGLVGEGPFPSMRAVVYRGILVVC IVLSTTIAIVDTVPDAWIGFEGIVSGAGGLF LVILSLDYILRLLVAWSQRAEGESGMAAL AGYALSPYGIFDFLAVVPFLVGEATALMPH DGETVFGILRFLKLARYSPALETLGVVVLH ELRPLLASLFIMLILAISASTVIYFVERAANP ALASVPAAMWWAIVTLSTVGFGDVVPITPLGKLFGSVVAVLGLCMFALPASILASGFAE EMKRQNFVSTWHLVAKVPFFQRLQASQIAEI AGLLKLSRAIKGEVLMREGDTGECMYFI VSGQVEVKGKAGTFILKNGDFFGEIALIE RCPRTATVKAVSRCQLLILDARDFHKFVAHD HALLEVIWETARSRMAQADKAHDAKEKEPV >mlK+ MSVLPFLRIYAPLNAVLAAPGLLAVAALTIPDMSGRSRLALAALLAVIWGAYLLQLAAT LLKRRAGVVRDRTPKIAIDVLAVLVPLAAF LLDGSPDWSLYCAVWLLKPLRDSTFFPVL GRVLANEARNLIGVTTLFGVVLFAVALAAYVIERDIQPEKFGSIPQAMWWAVVTLSTTG YGDTIPQSFAGRVLAGAVMMSGIGIFGLWAGILATGFYQEVRRGDFVRNWQLVAAVPL FQKLGPAVLVEIVRALRARTVPAGAVICRIGE PGDRMFFVVEGSVSVATPNPVELGPGAF FGEMALISGEPRSATVSAATTVSLLSLHSA DFQMLCSSSPEIAEIFR KTALERRGAAASA >hsCNGA1 MKLSMKNNIINTQQSFVTMPNVIVPDIEK EIRRMENGACSSFSEDDDSAYTSEESENENP HARGSFSYKSLRKGGPSQREQYLPGAIAIFNVNNSSNKDQEPEEKKKKKKEKKSKSDDK NENKNDPEKKKKKKDKEKKKKEEKSKDK KEHHKKEVVVIDPSGNTYYNWLFCITLPV MYNWTMVIARACFDELQSDYLEYWLILD YVSDIVYLIDMFVRTRTGYLEQGLLVKEEL KLINKYKSNLQFKLDVLSLI PTDLLYFKLGWNYPEIRLNR LLRFSRMFEFFQRTETRTNYP NIFRISNLVMYIVIIIHWNACVFYSISKAIGF GNDTWVYPDINDPEFG RLARKYVYSLYWS TLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFA TIVGNIGSMISNMNAARAEFQARIDAIK QYMHFRNVSKDMEKRVIKWFDYLWTNKKT VDEKEVLKYLPDKLRAEIAINVHLDTLK KVRIFADCEAGLLVELVLKLQPQVYSPGDY ICKKGDIGREMYIIKEGKLAVVADDGVTQ FVVLSDGSTFGEISILNIKGSKAGNRRTANIKSIGYSDLFCLSKDDLMEALTEYPDAKTML EEKGKQILMKDGLLDLNIANAGSDPKDLEEKVTRMEGSVDLLQTRFARILAEYESMQQ KLKQRLTKVEKFLKPLIDTEFSSIEGPWSESGPIDST >hsCNGA2 MTEKTNGVKSSPANNHNHHAPPAIKANG KDDHRTSSRPHSAADDDTSSELQRLADVDA PQQGRSGFRRIVRLVGIIREWANKNFREEEPRP DSFLERFRGPELQTVTTQEGDGKGDKD GEDKGTKKKFELFVLDPAGDWYYCWLFV IAMPVLYNWCLLVARACFSDLQKGYYLV WLVLDYVSDVVYIADLFIRLRTGFLEQGLLVKDTKKLRDNYIHTLQFKLDVASIIPTDLIY FAVDIHSPEVRFNRLLHFARMFEFFDRTETRT NYPNIFRISNLVLYILVIIHWNACIYYAIS KSIGFGVDTWVYPNITDPEYGYLAREYIYC LYWSTLTLTTIGETPPPV KDEEYLFVIFDFLI GVLIFATIVGNVGSMISNMNATRAEFQAKI DAVKHYMQFRKVSKGMEAKVIRWFDYL WTNKKTVDEREILKNLPAKLRAEIAINVHLS TLKKVRIFHDCEAGLLVELVLKLRPQVFS PGDYICRKGDIGKEMYIIKEGKLAVVADDGVT QYALLSAGSCFGEISILNIKGSKMGNRR TANIRSLGYSDLFCLSKDDLMEAVTEYPDAKKVLEERGREILMKEGLLDENEVATSMEV DVQEKLGQLETNMETLYTRFGRLLAEYTGAQ QKLKQRITVLETKMKQNNEDDYLSDG MNSPELAAADEP >hsCNGA3 MAKINTQYSHPSRTHLKVKTSDRDLNRAENGLSRAHSSSEETSSVLQPGIAMETRGLAD

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148 SGQGSFTGQGIARLSRLIFLLRRWAARHVHHQ DQGPDSFPDRFRGAELKEVSSQESNAQ ANVGSQEPADRGRSAWPLAKCNTNTSN NTEEEKKTKKKDAIVVDP SSNLYYRWLTAIA LPVFYNWYLLICRACFDELQSEYLMLWLVLDYSADVLYVLDVLVRARTGFLEQGLMVS DTNRLWQHYKTTTQFKLDVLSLVPTDLAYLKVGTNYPEVRFNRLLKFSRLFEFFDRTET RTNYPNMFRIGNLVLYILIIIHWNACIYFAISKF IGFGTDSWVYPNISIP EHGRLSRKYIYSL YWSTLTLTTIGETPPPVKDEEYLFVVVDFL VGVLIFATIVGNVGSMI SNMNASRAEFQAK IDSIKQYMQFRKVTKDLETRVIRWFDYLW ANKKTVDEKEVLKSLPDKLKAEIAINVHLD TLKKVRIFQDCEAGLLVELVLKLRPTV FSPGDYICKKGDIGKEMYIINEGKLAVVADDG VTQFVVLSDGSYFGEISILNIKGSKSGNRRTANIRSIGYSDLFCLSKDDLMEALTEYPEAK KALEEKGRQILMKDNLIDEELARAGADPKD LEEKVEQLGSSLDTLQTRFARLLAEYNAT QMKMKQRLSQLESQVKGGGDK PLADGEVPGDATKTEDKQQ >hsCNGA4 MEPEERRARKGWGHGFQETLGVEEWVWRG EAITKREEESVGIS LEVFSWAGVSPGPGR QGRVLELDCLHPFSSASSGCVAKSPKDKEVT AEAQQQPPSGSQALVPNSRSPLQAERVW TSHTPAPDHRTMSQDTKVKTTESSPPAPSKAR KLLPVLDPSGDYYYWWLNTMVFPVMY NLIILVCRACFPDLQHGYLVAWLVLDYTSD LLYLLDMVVRFHTGFLEQGILVVDKGRIS SRYVRTWSFFLDLASLMPTDVVYVRLGPH TPTLRLNRFLRAPRLFEAFDRTETRTAYPN AFRIAKLMLYIFVVIHWNSCLYFALSRYLGFGRDAWVYPDPAQPGFERLRRQYLYSFYF STLILTTVGDTPPPAREEEYLFM VGDFLLAVMGFATIMGSM SSVIYNMNTADAAFYPDH ALVKKYMKLQHVNRKLERRVIDWYQHLQI NKKMTNEVAILQHLPERLRAEVAVSVHL STLSRVQIFQNCEASLLEELVLKLQPQTYSP GEYVCRKGDIGQEMY IIREGQLAVVADDG ITQYAVLGAGLYFGEISIINIKGNMSGNRRTANIKSLGYSDLFCLSKEDLREVLSEYPQAQ TIMEEKGREILLKMNKLDVNAEAAEIALQEA TESRLRGLDQQLDDLQTKFARLLAELES SALKIAYRIERLEWQTREWPMPEDLAEADDEGEPEEGTSKDEEGRASQEGPPGPE >hsCNGB1 MLGWVQRVLPQPPGTPRKTKMQEEEEVEPE PEMEAEVEPEPNPEEAETESESMPPEESF KEEEVAVADPSPQETKEAALTSTISLRAQGA EISEMNSPSHRVLTWLMKGVEKVIPQPV HSITEDPAQILGHGSTGDTGCTDEPNEALEAQDTRPGLRLLLWLEQNLERVLPQPPKSSE VWRDEPAVATAPPGRPQEMGPKLQARETPSL PTPIPLQPKEEPKEAPAPEPQPGSQAQTS SLPPTRDPARLVAWVLHRLEMALPQPVLHGKIGEQEPDSPGICDVQTISILPGGQVEPDL VLEEVEPPWEDAHQDVSTSPQGTEVVPAY EEENKAVEKMPRELSRIEEEKEDEEEEEEEE EEEEEEEVTEVLLDSCVVSQVGV GQSEEDGTRPQSTSDQKL WEEVGEEAKKEAEEKAK EEAEEVAEEEAEKEPQDWAETKEEPEAEAEAASSGVPATKQHPEVQVEDTDADSCPLM AEENPPSTVLPPPSPAKSDTLI VPSSASGTHRKKLPSEDDEAE ELKALSPAESPVVAWSDP TTPKDTDGQDRAASTASTNSAIINDRLQEL VKLFKERTEKVKEKLIDPDVTSDEESPKPSP AKKAPEPAPDTKPAEAEPVEEEHYCDMLCCK FKHRPWKKYQFPQSIDPLTNLMYVLWL FFVVMAWNWNCWLIPVRWAFPYQTPDNIHHWLLMDYLCDLIYFLDITVFQTRLQFVRG GDIITDKKDMRNNYLKSRRFKMDLLSLLPLDFLYLKVGVNPLLRLPRCLKYMAFFEFNS RLESILSKAYVYRVIRTTAYLLYSLHLNS CLYYWASAYQGLGSTHWVYDGVGNSYIRC YYFAVKTLITIGGLPDPKTLFEIVFQLLNY FTGVFAFSVMIGQMRDVVGAATAGQTYYR SCMDSTVKYMNFYKIPKSVQNRVKTWYEYTWHSQGMLDESELMVQLPDKMRLDLAID VNYNIVSKVALFQGCDRQMIFDMLKRLRSVV YLPNDYVCKKGEIGR EMYIIQAGQVQV LGGPDGKSVLVTLKAGSVFGEISLLAVGGGNRRTANVVAHGFTNLFILDKKDLNEILVH YPESQKLLRKKARRMLRSNNKPKEEKSVLILPPRAGTPKLFNAALAMTGKMGGKGAKG GKLAHLRARLKELAALEAAAKQQELVEQAKS SQDVKGEEGSAAPDQHTHPKEAATDPP

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149 APRTPPEPPGSPPSSPPPASLGRPEGEEEGPAE PEEHSVRICMSPGPEPGEQILSVKMPEER EEKAE >hsCNGB3 MFKSLTKVNKVKPIGENNENEQSSRRNEEGSHPSNQSQQTTAQEENKGEEKSLKTKSTP VTSEEPHTNIQDKLSKKNSSGDLTTNPDPQNAAE PTGTVPEQKEMDPGKEGPNSPQNKP PAAPVINEYADAQLHNLVKRMRQRTALYK KKLVEGDLSSPEASPQTAKPTAVPPVKES DDKPTEHYYRLLWFKVKKMPLTEYLKRIKLPN SIDSYTDRLYLLWLLLVTLAYNWNCW FIPLRLVFPYQTADNIHYWLIADIICDIIYLY DMLFIQPRLQFVRGGDIIVDSNELRKHYRT STKFQLDVASIIPFDICYLFFGFNPMFRANRMLKYTSFFEFNHHLESIMDKAYIYRVIRTT GYLLFILHINACVYYWASNYEGIGTTRWVYDGEGNEYLRCYYWAVRTLITIGGLPEPQT LFEIVFQLLNFFSGVFVFSSLIGQMRDVIGAA TANQNYFRACMDDTIAYMNNYSIPKLVQ KRVRTWYEYTWDSQRMLDESDLLKTLPTTVQLALAIDVNFSIISKVDLFKGCDTQMIYD MLLRLKSVLYLPGDFVCKKGEIGKEMYIIK HGEVQVLGGPDGTKVLVTLKAGSVFGEIS LLAAGGGNRRTANVVAHGF ANLLTLDKKTLQEILVHYPD SERILMKKARVLLKQKAKT AEATPPRKDLALLFPPKEETPKLFKTLLGG TGKASLARLLKLKREQAAQKKENSEGGEE EGKENEDKQKENEDKQKENEDKGKENEDKDK GREPEEKPLDRPECTASPIAVEEEPHSV RRTVLPRGTSRQSLIISMAPSAEGGEEVLTIEVKEKAKQ >lpCNG MKRISIPTIRHNRETGKTVKYTPCSKHD LNLGDDLDEIAVISN AAPENVKYGTGSLTANG PRVVPGIITSTGMEHSRLVNEKRSSSSTRLPI SPPLYTTKSFENLKSKSASRQSLLETASSK LHGRDHKQRSRESITFSHKTHMGSELEISR KVREHRRKEIKREFEKWETSTSSSEKDGCN WTFVFDPSGSLSYYWSMIVSVAFLYNFWVVI FRFAFNEIKANTIVAWLTLDYFADILYAL DIVFHFRTGYLEEGVLQTDTIKLRHHYMNSTLFYIDCLCLLPIDFLYLSIGFNSILRCLRLV KIYRFWTFLDRTERHTNYPNVFRTVILTHYVLVIFHWNACLFHIISVNGGFGTRSWFNPR DETCNDVVCEYLRSFYWSTLALTTIGDLPSPRTKGEYLFLVVELVFGLFLFAAVLGHVA NIVTNVSTARKEFQARLDVVKTYMRMRRVP DHLQNKVIKWFDYLWVTQKSSDEDRSV GFLPDKLKAEIAIHVHLNTLKQVEIFQNTEAGFLCELVLRLRPVLFSPGDYICRKGEVGKE MYIVNRGRLEVVTDNGKTVLATLRAGSYFG EISILNMSTTGNRRTASVRSVGYSDLFCL NKQDMWDVLKDYPAARDRLESIAVKRLEKYR KDPLQNIAMSRSHSTPGLLESHGQVPL ENMPLYPREPSTTASAATLLSDDECRQNVRATASGTRSRRTSVDDIQQRGTPLYEQIEPT LRSSFPTHTGAQRSGGRLYLVESSLDRQLVA TTTSHTDLFTPLPPSPY FVHSSSHNTFDVS PVSDYSAMSSTGVDRTIYSSHSPHVPVPSPI YVSHIGRVTPHSDRDSHPESLVTEIKRLRER LMTLETENASMGMKLSHQQWEVENRLAEIEL QVCGTGGETSPISCGSTDENERNRESVI >dmCNG MRHFKVKAMVQSLDISAITGQQTDAEPSKRSKPSALRRTLQALRQRLTKRNRPKPPDWF LEKFSNTTNTDKIGKGCPAMEDAALSSEIRGSSVLCNRLSVDPTLQSHYRWLAIVSLAVL YNIIFVVGRAVFWEINKSAPAFWYTLDYLCDFIYLLDTLVHMHEGFLDQGLLVRDAFRL RRHYFHTKGWYLDVLSMLPTDLAYIWWPPETCSSLYLPCPVIVRLNRLLRINRLWEWFD RTETATGYPNAFRICKVVLAILVLIHWNACMYFAISYEIGFSSDSWVYNLNGTRNNTLQR QYIYSFYWSTLTLTTIGETPTPENDVEYLF VVADFLAGVLIFATIVGNIGSMISNMNVARV EFQNRMDGVKQYMAFRRV GHELEARVIRWFAYTWSQSG ALDEERVLAALPDKLKAEI AIQVHMDTLKQVRIFHDTEPGLLEALVLKLK LQVFSPGDYICRKGDVGKEMYIVKRGKL SVVGDDGITVLATLGAGSVFGEVSVLEIAGNRTGNRRTANVRSLGYSDLFCLAKRDLWE TLSDYPEARSTLTQRGCQLLRKDGLLDEQIF ADSQRVHDSIEGGIE KLELSVENLNMRLA RLLAEYTASQAKIKQRLAKLEMNGGPGTWRLECEPQSRARSGRLYSLQPKRRPRSRPDA TAKSSDAAKQNTL

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150 >ceCNG1 MYQVPKRAKTNLAREIRKREFSYVDRQKASK PTQLSEKEWKSPRSEDSFDLLDPANASK EPSASTRPLPYPPTRPPEVVIQIDEVESPILG LIDETDDHELDGRLDPASSFDANSLSATRAS SIIEDDVRSQISFIMRERLHSIAKEVHRRTSAVREDLIRETPEDTVSMASNVPKQNEHRPSL MSLIGLQNRSESPTVDTVKNCFGFSLKGTF HPYGRFYMTWLSLVTLCFLFNAFCIPLRSS YPYQTADNWMYWFIVDYSCDLVYVIDMLL IKPRLRFTRGGIQVKIYKDTQRHYLMTRT FKLDILSILPTDLMYFFFGKMPIWRINRVLKINSFWLLFDMLDNSFANPYAIRIARTLSYM IYIIHCNSCVYYKLSALQAFGQIAYLENGKWY LNKWVYNNQGNAYIRCFYFTAAVATST GNNPAPTNVIEYIYMTCSWMMGVFVFALLLGQIRDIVSNANRNREEFQRKMDLALGEC KKLGLKMETTNRVRDWFIYTWQQQKTLDE KKLIEKLPLKLQTDLALSVHYTTLSKVQL FQDCDRALLRDLVLKLRPVIFLPGDMICL KGDVGKEMYIINQGIL QVVGGDHNEKIFAEL AQGAVFGEISLLAIGGNNRRTASIRAKGYCTLF VLAKEDLNDVIRYYP QAQTILRRKAAA MLKNDKKSDEKTEKIKAQAELEDRCKINPR QVPKLITLIANMTEMNENKGVQELKKVIE EETEKSRRQSIYYPWSTLQRDDD DEEEWNDEEDLSDVGEDF DLDPTNHSDDEDPMEDV DLAPEVHDDDWDQPGTSGTQKLHAD >ceCNG2 MSTAEPAPDPTNPSTSGLAPTTNGIGSPPPTA SAATKFSILTKFLRR KNQVHTTTAQQNEF MQKYMPNGNSNAVQPAATGGQPASSDGGSAI EVPPPKESYAVRIRKYLANYTQDPSTD NFYYWTCVVTVAYIYNLLFVIARQVFNDLIGPSSQSLCRFYNGTLNSTTQVECTYNMLT NMKEMPTYSQYPDLGWSKYWHFRMLWVFFDLLMDCVYLIDTFLNYRMGYMDQGLV VREAEKVTKAYWQSKQYRIDGISLIPLDYILG WPIPYINWRGLPILRLNRLIRYKRVRNCL ERTETRSSMPNAFRVVVVVWYIVIIIHWNAC LYFWISEWIGLGT DAWVYGHLNKQSLPD DITDTLLRRYVYSFYWSTLILTT IGEVPSPVRNIEYAFVTLDLMCGVLIFATIVGNVGSMIS NMSAARTEFQNKMDGIKQYMELRKVSKQ LEIRVIKWFDYLWTNKQSLSDQQVLKVLP DKLQAEIAMQVHFETLRKVRIFQDCEAGL LAELVLKLQLQVFSP GDFICKKGDIGREMYI VKRGRLQVVDDDGKKVFVTLQEGSVFGEL SILNIAGSKNGNRRTANVRSVGYTDLFVL SKTDLWNALREYPDARKLLLAKGREILKK DNLLDENAPEEQKTVEEIAEHLNNAVKVL QTRMARLIVEHSSTEGKLMKRIEMLEKHLSR YKALARRQKTMHGVS IDGGDISTDGVDE RVRPPRLRQTKTIDLPTGTESESLLK >spCNG MPRGKKPDGLEMNNLQLKPGETSPRSPLTPTIKLSRPPSSSIERKSSLSYMFVNANEEADL VNGGFITNDKVSNNQQHQHQ NGNGAADASRGANSAPPCNGHSPVESWTQSVSPASNHS NGDVHNSHNHKNKNGAY SISKCLMPPTKRSKSKRHSLPLPGFVESSSLKSSDDEADAPQ RITLGNGGSTHSVNLLPHSMPSPTKSNAS PWKRWFKRKPSNAGSEQSSFSDASWMSSAE YDDADNKKPKLWNLARSITKRVSKLKNNE VAPMPQNTLAPPEPTATADDSLKPTSTRR RLDSFAPSSIGDVAEEIHFKTSRFGLITRCW ERIKSVRFPEYFDPLGGLWLFWLSVVTMAF LYNAIVIFLRGAFQEKYQNKNNLGYWLTCDYL CDFIYIIDMLLFRARLMFFQAGQGIFDL FSLLPLDLFYFSLNKVEPLLRLPRLFKFHSYLEFSQKFENKSKSAHGIRIFRMVMYLLYMI HLNTCLYYAILFWEGIRQDDIAMANDYW VYLDPKVDVYTKCLFRSAKTLIVIGNLPPPN TNSQIIFMNIDFIVGVFVFASMIGQMRDIS AKAGATKDQFRRQMDDTMSLLQVWKIPEA VQKRVRTYYIYSWDQGAVLDERDLLLGVPI RMQTDIAINVHMDTLSRVSLFQDCDKML LRDLVLKLRPLVYLPNEYICRKGAVGKEMY IVVDGSVQVLGGDKKVLATLQPGSVFGEI SLLSVGHGNRRTADVAAPGFANLFV LDKKDLQEVVVNYPDAQESLKRKAR >osCNG MRLNLIENEARHRQNNLATGSVKTMSSKIVLKLKDFTRIWISREESMLDPGGNVVLMW NRVFLVSCVASHFIDPLFFFLPIVERRDRQLC MTMDHHLAIILTCLRSFLDIFFIAHIAISFS

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151 TAHVDPSSKVLGRGELVTDPK KIANRYIRTNFFIDLVAALPVPQVLVWIAMPSISFKHINA PFFLIILVQSAIRLYIVILLSLSIMEMVGFIAKN GWEGAIYSLVLYLVASHVVGAIFYLTAV DRQKTCWETQCSIEDRMAHKGLCDLHFLDCKYATSSNSQSWANSTNVFTHCNANSNSV SINYGIFIQAIQNGVTTASFSEKYFYSLCTY GNPLVTSSFIGENLFAIGLTLLSIGLFAQLIG NMQIHMRSLSKNTEDWRMWQTEMEDWM IDHQIPDELRYRISQFFKYKWFATQGVEED SILRQLPADLHRDIKRYLCLDLVERVPFFSAM DHQLLDAICERMTYFLRTEGTYITREGD PVKVMLFIIRGKLESSTTDGGRTGFFNSIILKPGDFCGEELLTWALLPSSRDSYPSSTRTVK TIAELEAFSLQADDIKCVASTFRMMHSKHL QHTFRLHSYQWRTWAARFIQSAWRRRQN RQKMAEVGLSNRWKSFFSLVNDFNDTRCEDINGS SSTVSHRETVTVSKIASIFKKAQKER PEEPDFSEDHHPE >atCNG1 MNFRQEKFVRFQDWKSDKTSSDVEYSGKNEIQTGIFQRTISSISDKFYRSFESSSARIKLF KRSYKSYSFKEAVSKGIGSTHKILDPQGPFL QRWNKIFVLACIIAVSLDPLFFYVPIIDDAK KCLGIDKKMEITASVLRSFTDVFYVLHIIF QFRTGFIAPSSRVFGRGVLVEDKREIAKRYL SSHFIIDILAVLPLPQMVILIIIPHMRGSSSLNTKNM LKFIVFFQYIPRFI RIYPLYKEVTRTS GILTETAWAGAAFNLFLYMLAS HVFGAFWYLFSIERETVCWKQA CERNNPPCISKLLYC DPETAGGNAFLNESCPIQTPNTTLFDFGIFLDALQSGVVESQDFPQKFFYCFWWGLQNLS SLGQNLKTSTYIWEICFAVFISIAGLVL FSFLIGNMQTYLQST TTRLEEMRVKRRDAEQW MSHRLLPENLRKRIRRYEQ YKWQETRGVDEENLLSNLPKDL RRDIKRHLCLALLMRVP MFEKMDEQLLDALCDRLQPVLYTEESYIVR EGDPVDEMLFIMRGKLLTITTNGGRTGFL NSEYLGAGDFCGEELLTWALDPHSSSNLPISTRTVRALMEVEAFALKADDLKFVASQFR RLHSKQLRHTFRYYSQQWKTWAACFIQAAWRRYIKKKLEESLKEEENRLQDALAKEAC GSSPSLGATIYASRFAANILRTIRRSGSV RKPRMPERMPPMLLQKPAEPDFNSDD >atCNG2 MPSHPNFIFRWIGLFSDKFRRQTTGIDENSN LQINGGDSSSSGSDETPVLSSVECYACTQV GVPAFHSTSCDQAHAPEWRASAGSSLVPIQ EGSVPNPARTRFRRLKGPFGEVLDPRSKR VQRWNRALLLARGMALAVDPLFFYALSIGRTT GPACLYMDGAFAAVVTVLRTCLDAV HLWHVWLQFRLAYVSRESLVVGCGKLVWDPRAIASHYARSLTGFWFDVIVILPVPQAV FWLVVPKLIREEKVKLIMTILLLIFLFQFLPK IYHCICLMRRMQKVTGYIFGTIWWGFALN LIAYFIASHVAGGCWYVLAIQRVASCIRQQC MRTGNCNLSLACKEEVCYQFVSPTSTVG YPCLSGNLTSVVNKPMCLDSNGPFRYGIYRWA LPVISSNSLAVKILYPIFWGLMTLSTFA NDLEPTSNWLEVIFSIVMVL SGLLLFTLLIGNIQVFLHAV MAKKRKMQIRCRDMEWWM KRRQLPSRLRQRVRRFERQRWNALGGED ELELIHDLPPGLRRDIKRYLCFDLINKVPLFR GMDDLILDNICDRAKPRVFSKDEKIIREGDP VQRMIFIMRGRVKRIQSLSKGVLATSTLEP GGYLGDELLSWCLRRPFLDRLPPSSATFVCLENIE AFSLGSEDLRYITDHFRYKFANERL KRTARYYSSNWRTWAAVNIQM AWRRRRKRTRGENIGGSMSPVSENSIEGNSERRLLQY AAMFMSIRPHDHLE >atCNG3 MMNPQRNKFVRFNGNDDEFSTKTTRPSVSSV MKTVRRSFEKGSEKIRTFKRPLSVHSNK NKENNKKKKILRVMNPNDSYLQSWNKIFLLLS VVALAFDPLFFYIPYVKPERFCLNLDK KLQTIACVFRTFIDAFYVVHMLFQFHTGF ITPSSSGFGRGELNEKHKDIALRYLGSYFLID LLSILPIPQVVVLAIVPRMRRPASLVAKELLKWVI FCQYVPRIARIYPLFKEVTRTSGLVTE TAWAGAALNLFLYMLASHVFGSFWYLISI ERKDRCWREACAKIQNCTHAYLYCSPTGE DNRLFLNGSCPLIDPEEITNSTVFNFGIFA DALQSGVVESRDFPKKFFYCFWWGLRNLSA LGQNLKTSAFEGEIIFAIVICISGLVLFAL LIGNMQKYLQSTTVRVEEMRVKRRDAEQWM SHRMLPDDLRKRIRKYEQYKWQETKGVEEEA LLSSLPKDLRKDIKRHLCLKLLKKVPW

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152 FQAMDDRLLDALCARLKTVLYTEKSYIV REGEPVEDMLFIMRGNLISTTTYGGRTGFFN SVDLVAGDFCGDLLTWALDPLSSQFPISSRTVQALTEVEGFLLSADDLKFVATQYRRLH SKQLRHMFRFYSVQWQTWAACFIQAAWKRHCRRKLSKALREEEGKLHNTLQNDDSGG NKLNLGAAIYASRFASHALRN LRANAAARNSRFPHMLTLLPQKPADPEFPMDET >atCNG4 MATEQEFTRASRFSRDSSSVGYYSEED NTEEEDEEEEEMEEIEEEEEEEEEEDPRIGLTCG GRRNGSSNNNKWMMLGRILDPRSKWVREWNKVFLLVCATGLFVDPLFLYTLSVSDTC MCLLVDGWLALTVTALRSMTDLLHLWNIWI QFKIARRWPYPGGDSDGDTNKGGGTRG STRVAPPYVKKNGFFFDLFVILPLPQVVLWVVIPSLLKRGSVTLVVSVLLVTFLFQYLPKI YHSIRHLRRNATLSGYIFGTVWWGIALNMIAYFVAAHAAGACWYLLGVQRSAKCLKEQ CENTIGCDLRMLSCKEPVYYGTTVMVLD RARLAWAQNHQARSVCLDINTNYTYGAYQ WTIQLVSSESRLEKILFPIFWGLMTLSTFGN LESTTEWSEVVFNIIVLTSGLLLVTMLIGNI KVFLHATTSKKQAMHLKMRNIEWWMKKRHLPIGFRQRVRNYERQRWAAMRGVDECE MVQNLPEGLRRDIKYHLCLDLVRQVPLFQHMDDLVLENICDRVKSLIFTKGETIQKEGD AVQRMLFVVRGHLQSSQLLRD GVKSCCMLGPGNFSGDELLSWCLRRPFVERLPPSSSTL VTLETTEAFGLDAEDVKYVTQHFRYTFVNE KVKRSARYYSPGWRTWAAVAVQLAWR RYKHRLTLTSLSFIRPRRPLSRCASLGEDKLRLYAAILTSPKPNPDDFDDY >atCNG5 MAGKRENFVRVDDLDSRLPSSSVAFQQNYA SNFSGQLHPIHASNETSRSFKKGIQKGSK GLKSIGRSLGFGVYRAVFPEDLKVSEKKIFD PQDKFLLYCNKLFVAS CILSVFVDPFFFYL PVINAESKCLGIDRKLAITASTLRTFIDVFYLAHMALQLRTAYIAPSSRVFGRGELVIDPA QIAKRYLQRWFIIDFLSVLPLPQIVVWRFLQSSNGSDVLATKQALLFIVLVQYIPRFLRVL PLTSELKRTAGVFAETAWAGAAYYLLLY MLASHIVGAFWYLLALERNDACWQEACID AGNCSTDFLYCGNQNMDGYAVWNRAKESVL KSKCRADLDDNNPPFDFGIYTQALSSGI VSSQNFIVKYCYCLWWGLQNLSTLGQGLETS TYPMEIIFSISLAISGLILFALLIGNMQTYL QSLTIRLEEMRVKRRDSEQWMHHRMLPQDLRERVRRYDQYKWLETRGVDEEYLVQNL PKDLRRDIKRHLCLALVRRVPLFKSMDDKLLDAICMRLKPCLFTESTYLVREGDPVDEM LFIIRGRLESVTTDGGRSGFFNRSLLKEG EFCGEELLTWALDPKSGVNLPSSTRTVKALTE VEAFALTSEELKFVASQFRR LHSRQVQHTFRFYSHQWRTWAACFIQAAWRRYCKRKK MEEAEAEAAAVSSSTAGPSYSIGAAFLATKF AANALRTIHRNRNTKIRDLVKLQKPPEPD FTAD >atCNG6 MFDTCGPKGVKSQVISGQRENFVRLDSMDSR YSQSSETGLNKCTLNIQGGPKRFAQGSK ASSGSFKKGFRKGSEGLWSIGRSIGLGVSRA VFPEDLEVSEKKIFDPQDKFLLLCNKLFV ASCILAVSVDPLFLYLPFINDKAKCVGIDRKL AIIVTTIRTVIDSFYLFHMALRFRTAYVAP SSRVFGRGELVIDPAQIAKRYLQQYFIIDLLS VLPVPQIIVWRFLYTSRGANVLATKQALR YIVLVQYIPRFLRMYPLSSELKRTAGVFA ETAWAGAAYYLLLYMLASHIVGALWYLLA LERNNDCWSKACHNNQNCTRNFLFCGN QNMKGYAAWDNIKVSYLQLKCPVNVPEDE EPPFDFGIYLRALSSGIVSSKNFVSKYFFCLW WGLQNLSTLGQGLETSTYPGEVIFSITLAI AGLLLFALLIGNMQTYLQSLTI RLEEMRVKRRDSEQWMHHR MLPPELRERVRRYDQYK WLETRGVDEENLVQNLPKDLR RDIKRHLCLALVRRVPLFENMDERLLDAICERLKPCLF TEKSYLVREGDPVNEMLFIIRGRLESVTTD GGRSGFYNRSLLKEGDFCGDELLTWALDP KSGSNLPSSTRTVKALTEVEAFALIADELK FVASQFRRLHSRQVQHTFRFYSQQWRTWA ACFMQAAWRRYIKRKKLEQLRKEEEEEEAAAA SVIAGGSPYSIRATFLASKFAANALRS VHKNRTAKSTLLLSSTKELV KFQKPPEPDFSAEDH >atCNG7

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153 MMMQRNCFGFNLKNRGGEKKKAS KSFREGVKKIRSEGLITI GKSVTRAVFPEDLRITEK KIFDPQDKTLLVWNRLFVISCILAVSVDPLFF YLPIVDNSGSSCIGID TKLAVTTTTLRTIV DVFYLTRMALQFRTAYIAPSSRVFGRGELVIDPAKIAERYLTRYFVVDFLAVLPLPQIAV WKFLHGSKGSDVLPTKTALLNIVIVQYIPRFVRFIPLTSELKKTAGAFAEGAWAGAAYYL LWYMLASHITGAFWYMLSVERNDTCWRFACKVQPDPRLCVQILYCGTKFVSSGETEWI KTVPELLKSNCSAKADDSKFNYGIYGQAISSG IVSSTTFFSKFCYCLWWGLQNLSTLGQG LQTSTFPGEVLFSIAIAIAG LLLFALLIGNMQTYLQSLTVR LEEMRIKRRDSEQWMHHRSL PQNLRERVRRYDQYKWLETRGVDEENIVQS LPKDLRRDIKRHLC LNLVRRVPLFANMD ERLLDAICERLKPSLFTESTYIVREGDPVNEMMFIIRGRLESVTTDGGRSGFFNRGLLKEG DFCGEELLTWALDPKAGSNLPSSTRTVKAL TEVEAFALEAEELKFVASQFRRLHSRQVQ QTFRFYSQQWRTWASCFIQAAWRRYSRRKNAELRRIEEKEEELGYEDEYDDESDKRPM VITRSESSSRLRSTIFASRFAANALKGH RLRSSESSKTLINLQKPPEPDFDAE >atCNG8 MSSNATGMKKRSCFGLFNVTSRGGGKTKNTSKSFREGVKIGSEGLKTIGKSFTSGVTRA VFPEDLRVSEKKIFDPQDKTLLLWNRMFVISCILAVSVDPLFFYLPIVDNSKNCIGIDSKL AVTTTTLRTIIDVFYLTRMALQ FRTAYIAPSSRVFGRGELVIDPAKIAERYLTRYFIVDFLA VLPLPQIAVWKFLHGSKGTDVLPTKQALLHIVITQYIPRFVRFIPLTSELKKTAGAFAEGA WAGAAYYLLWYMLASHITGAFWYMLSVERNDTCLRSACKVQPDPKVCVQILYCGSKL MSSRETDWIKSVPDLFKNNCSAKSDESKFN YGIYSQAVSSGIVSSTTFFSKFCYCLWWGL QNLSTLGQGLQTSTYPGEVLFSIAIAVAGLLLFALLIGNMQTYLQSLTVRLEEMRIKRRD SEQWMHHRSLPQNLRERVRRYDQYKWLETR GVDEENIVQSLPKDLRRDIKRHLCLNLV RRVPLFANMDERLLDAICERLKPSLYTESTYIVREGDPVNEMLFIIRGRLESVTTDGGRSG FFNRGLLKEGDFCGEELLTWALDPKAGSNL PSSTRTVKALTEVEAFALEAEELKFVASQ FRRLHSRQVQQTFRFYSQQWRTWAAC FIQAAWRRHLRRKIAELRRKEEEEEEMDYEDD EYYDDNMGGMVTRSDSSVGSSSTLRSTVFASRFAANALKGHKLRVTESSKSLMNLTKP SEPDFEALDTDDLN >atCNG9 MLDCGKKAVKSQVISGRLEKFVRLDSMDSR YSQTSDTGLNRCTLNLQGPTRGGGAQGN NVSSGSFKKGFRKGSKGLWS IGRSIGLGVSRAVFPEDL KVSEKKIFDPQDKFLLLCNKLF VTSCILAVSVDPLFLYLPFVKDNEKCIGIDRK LAIIATTLRTVIDAFYLFHMALRFRTAFV APSSRVFGRGELVIDPAQIAK RYLQQYFIIDFLSVLPLPQI VVWRFLYISKGASVLATKRA LRSIILVQYIPRFIRLYPLSSELKRTAGVF AETAWAGAAYYLLLYML ASHIVGAIWYLLAL ERYNGCWTKVCSNSSLDCHRNFLFCGNEKM DGYAAWTTIKDSVLQLNCPVNTTDNPPF DFGIYLRALSSGIVSSKSFVSKYFFCLWWGL QNLSTLGQGLETSTYPGEVIFSIALAIAGL LLFALLIGNMQTYLQSLTIRLEEMRVKRRD SEQWMHHRMLPPELRERVRRYDQYKWLE TRGVDEENLVQNLPKDLRRDIKRHLCLALVRRVPLFENMDERLLDAICERLKPCLYTES SYLVREGDPVNEMLFIIRGRLESVTTDGGR SGFFNRSLLKEGDFCGEELLTWALDPKSGS NLPSSTRTAKALTEVEAFALIADELKFVASQFRR LHSRQVQHTFRFYSQQWRTWAAIFIQ AAWRRYVKKKKLEQLRKEEEEGEGSVTSIR ATFLASKFAANALRKVHKNRIEAKSTIEL VKYQKPSEPDFSADDTS >atCNG10 MAFSHDNRVRFKDEGKPLSSEYGYGRKAR PSLDRVFKNVKWGFKKPLSFPSHKDPDHK ETSSVTRKNIINPQDSFLQNWNKIFLFACVVA LAIDPLFFYIPIVDSARHCLTLDSKLEIAA SLLRTLIDAFYIIHIVFQFRTAYIAPSSRVFGR GELVDDAKAIALKYLSSYFIIDLLSILPLPQ IVVLAVIPSVNQPVSLLTKDYLKFSIIAQYVP RILRMYPLYTEVTRTSGIVTETAWAGAA WNLSLYMLASHVFGALWYLISVEREDRCWQEACEKTKGCNMKFLYCENDRNVSNNFL

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154 TTSCPFLDPGDITNSTIFNFGIFTDALKSGVVESHDFWKKFFYCFWWGLRNLSALGQNLQ TSKFVGEIIFAISICISGLVLFALLIGNMQ KYLESTTVREEEMRVRKRDAEQWMSHRMLP EDLRKRIRRYEQYRWQETR GVEEETLLRNLPKDLRRDIKRHLC LDLLKKVPLFEIMDEQ LLDAVCDRLRPVLYTENSYVIREGDPVGEMLFVMRGRLVSATTNGGRSGFFNAVNLKA SDFCGEDLLPWALDPQSSSHFPISTRTVQ ALTEVEAFALTAEDLKSVASQFRRLHSKQLQ HTFRFYSVQWRTWSVSFIQAAWRRYCRR KLAKSLRDEEDRLREALASQDKEHNAATVS SSLSLGGALYASRFASNALHNLRHNISNLPPRYTLPLLPQKPTEPDFTANHTTDP >atCNG11 MNLQRRKFVRLDSTGVDGKLKSVRGRLK KVYGKMKTLENWRKTVLLACVVALAIDPL FLFIPLIDSQRFCFTFDKTLVAVVCVIRTFID TFYVIHIIYYLITETIAP RSQASLRGEIVVHS KATLKTRLLFHFIVDIISVLPIPQVVVLTLIPLSASLVSERILKWIILSQYVPRIIRMYPLYKE VTRAFGTVAESKRVGAALNFFLYMLHSYVCGAFWYLSSIERKSTCWRAACARTSDCNL TVTDLLCKRAGSDNIRFLNTSCPLIDPAQITNSTDFDFGMYIDALKSGVLEVKPKDFPRKF VYCFWWGLRNISALGQNLETS NSAGEIFFAIIICVSGLLLF AVLIGNVQKYL QSSTTRVDE MEEKKRDTEKWMSYREIPEYLKERIRRF EDYKWRRTKGTEEEALLRSLPKDLRLETKRY LFLKLLKKVPLLQAMDDQLLDALCARLKTVHYTEKSYIVREGEPVEDMLFIMRGNLIST TTYGGRTGFFNSVDLIAGDSCGDLLTWALYSLSSQFPISSRTVQALTEVEGFVISADDLKF VATQYRRLHSKQLQHMFRFYSLQWQTWAAC FIQAAWKRHCRRKLSKALREEEGKLHN TLQNDDSGGNKLNLGAAIYA >atCNG12 MNHRRSKFARIDSMGVDGKLKSVRGRLK KVYGKMKTLENWRKTVLLACVVALAIDPL FLFIPLIDSQRFCFTFDKTLVAVVCVIRTFID TFYVIHIIYYLITETIAP RSQASLRGEIVVHS KATLKTRLLFHFIVDIISVLPIPQVVVLTLIPLSASLVSERILKWIILSQYVPRIIRMYPLYKE VTRAFGTVAESKWAGAALNLFLYMLHSYVFGAFWYLSSIERKSKCWRAACARTSDCN LTVTDLLCKRAGSDNIRFLNTSCPLIDPAQITNSTDFDFGMYIDALKSGVLEVKPKDFPRK FVYCFWWGLRNISALGQNLETS NSAGEIFFAIIICVSGLLLF AVLIGNVQKYL QSSTTRVD EMEEKRRDTEKWMSYRVIPEY LKERIRRFEDYKWRETKG TEEEALLRSLPKDLRLETKR YLYLDMLKRVPWLNIMDDGWLLEAVCDRVKSVFYLANSFIVREGHPVEEMLIVTRGKL KSTTGSHEMGVRNNCCDLQDGDICGELLFNGSR LPTSTRTVMTLTEVEGFILLPDDIKFI ASHLNVFQRQKLQRTFRLYSQQWRSWAAFFIQAAWRKHCKRKLSKTRDNENIPQGTQL NLASTLYVSRFVSKALQNRRKDTADCSSSPDMSPPVPHKPADLEFAKAEA >atCNG13 MEFKRDNTVRFYGDEKQTIEVGEKRVPLFKSTTAPFMKQEVLPKKSKTRLKIPRFGRFK VFPENFEIERDKILDPGGDAVLQWNRVFLFWCLVALYVDPLFFFLSSVKRIGRSSCMTTD LKLGIVITFFRTLADLFYVLHIVIKFRTAYVSR TSRVFGRGELVKDPK LIARRYLRSDFIVD LIACLPLPQIVSWFILPSIRSSHSDHTTNALVLIVLVQYIPRLYLIFPLSAEIIKATGVVTTTA WAGAAYNLLQYMLASHILGSAWYLLSIERQATCWKAECHKESVPLQCVTDFFDCGTLH RDDRNNWQNTTVVFSNCDPSNNIQFTFGIFADALTKNVVSSPFLEKYLYCLWFGLQNLS SYGQNLSTSTSVLETMFAILVAIFGLVLFALLIGNMQTYLQSITVRLEEWRLKRRDTEEW MGHRLLPQNLRERVRRFVQYKWLATRGVDEETI LHSLPADLRRDIQR HLCLDLVRRVPL FAQMDDQLLDAICERLASSLSTQGNYIVRE GDPVTEMLFIIRGKLESSTTNGGRTGFFNSI TLRPGDFCGEELLAWALLPKSTVNLPSSTRTVRALEEVEAFALQAGDLKFVANQFRRLH SKKLQHTFRYYSHQWRTWAACFVQVAWRRYKRKKLAKSLSLAESFSSYDEEEAVAVA ATEEMSHEGEAQSGAKARHHT SNVKPHFAATILASRFAKNTRRTAHKLKDVEIPMLPKP DEPDFSVDD >atCNG14

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155 MEFKRDNTVRFYGDEKQTIEVGEKRVPLFKSTTAPFMKQEVLPKKSKTRLKIPRFGRFK VFPENFEIERDKILDPGGDAVLQWNRVFLFWCLVALYVDPLFFFLSSVKRIGRSSCMTTD LKLGIVITFFRTLADLFYVLHIVIKFRTAYVSR TSRVFGRGELVKDPK LIARRYLRSDFIVD LIACLPLPQIVSWFILPSIRSSHSDHTTNALVLIVLVQYIPRLYLIFPLSAEIIKATGVVTTTA WAGAAYNLLQYMLASHILGSAWYLLSIERQATCWKAECHKESVPLQCVTDFFDCGTLH RDDRNNWQNTTVVFSNCDPSNNIQFTFGIFADALTKNVVSSPFLEKYLYCLWFGLQNLS SYGQNLSTSTSVLETMFAILVAIFGLVLFALLIGNMQTYLQSITVRLEEWRLKRRDTEEW MGHRLLPQNLRERVRRFVQYKWLATRGVDEETI LHSLPADLRRDIQR HLCLDLVRRVPL FAQMDDQLLDAICERLASSLSTQGNYIVRE GDPVTEMLFIIRGKLESSTTNGGRTGFFNSI TLRPGDFCGEELLAWALLPKSTVNLPSSTRTVRALEEVEAFALQAGDLKFVANQFRRLH SKKLQHTFRYYSHQWRTWAACFVQVAWRRYKRKKLAKSLSLAESFSSYDEEEAVAVA ATEEMSHEGEAQSGAKARHHT SNVKPHFAATILASRFAKNTRRTAHKLKDVEIPMLPKP DEPDFSVDD >atCNG15 MGYGNSRSVRFQEDQEVVHGGESGVKLK FKINGTQINNVKMMSKGKFLKAKVLSRVF SEDLERVKTKILDPRGQTIRRWNKIFLIACL VSLFVDPLFFFLPVMRNEACITIGVRLEVVL TLIRSLADAFYIAQILIRFRTAYIAPPSRVFGR GELVIDSRKIAWRYLHKSFWIHLVAALPL PQVLIWIIIPNLRGSPMTNTKNVLRFIIIFQYVP RMFLIFPLSRQIIKATGVVTETAWAGAA YNLMLYMLASHVLGACWYLLAVERQEACWRHACNIEKQICQYRFFECRRLEDPQRNS WFEWSNITTICKPASKFYEFGIFGDAVTST VTSSKFINKYFYCLWWGLKNLSSLGQNLAT STYAGEILFAIIIATLGLVLF ALLIGNMQTYLQSTTMRLEE WRIRRTDTEQWMHHRQLPP ELRQAVRKYDQYKWLATRGVDEEALLISLPLD LRRDIKRHLCFDLVRRVPLFDQMDER MLDAICERLKPALCTEGTFLVREGDPVNEMLFIIRGHLDSYTTNGGRTGFFNSCLIGPGD FCGEELLTWALDPRPVVILPSSTRTVKAICE VEAFALKAEDLQFVASQFRRLHTKQLRHK FRFYSHQWRTWAACFIQAAWRRHRKRKYK TELRAKEEFHYRFEAATARLAVNGGKYT RSGSDSGMMSSIQKPVEPDFSSE >atCNG16 MSNLHLYTSARFRNFPTTFSLRHHHNDPNNQRRRSIFSKLRDKTLDPGGDLITRWNHIFLI TCLLALFLDPLYFYLPIVQAGTACMSIDVRFGIFVTCFRNLADLSFLIHILLKFKTAFVSKS SRVFGRGELVMDRREIAIRYLKSEFVIDL AATLPLPQIMIWFVIPNAGEFRYAAHQNHTLS LIVLIQYVPRFLVMLPLNRRIIKATGVAAKT AWSGAAYNLILYLLVSHVLGSVWYVLSIQ RQHECWRRECIKEMNATHSPSCSLLFLDCG SLHDPGRQAWMRITRVLSNCDARNDDDQ HFQFGMFGDAFTNDVTSSPFFD KYFYCLWWGLRNLSSYGQSLAASTLSSETIFSCFICVA GLVFFSHLIGNVQNYLQSTTARLDEWRVR RRDTEEWMRHRQLPDELQERVRRFVQYK WLTTRGVDEEAILRALPLDLR RQIQRHLCLALVRRVPFFAQMDDQLLDAICERLVPSLNT KDTYVIREGDPVNEMLFIIRGQMESSTTDGG RSGFFNSITLRPGDFCGEELLTWALVPNIN HNLPLSTRTVRTLSEVEAFALRAEDLKFVANQFRRLHSKKLQHAFRYYSHQWRAWGTC FIQAAWRRYMKRKLAMELARQEEEDDYF YDDDGDYQFEEDMPES NNNNGDENSSNN QNLSATILASKFAANTKRGVLGNQRGSTRIDPDHPTLKMPKMFKPEDPGFF >atCNG17 MELRKDKLLMFYSEGKESKEAKWAVNDPMSKSYKLSLPSALRPDNLLPGNRLRYTDAS KSKSSKVSWYKTILDPGSEIVLKWNWVFIV SCMVALFIDPLYFFVPAIGGDKNYPCARTD TSLSILVTFFRTIADLFYLLHIFIKFRTGFIAPNSSTRVFGRGELVMDPKAIAWRYIKSDFII DLIATLPLPQIVIWFVISTTKSYRFDHNNNAI ALIVLLQYIPRFYLIIPLSSQIVKATGVVTK TAWAGAAYNLLLYMLASHVLGAAWYILSVDR YTSCWKSRCNGEAGQVNCQLYYLDC DSMYDNNQMTWANVTKVFKL CDARNGEFKYGIFGNAITKNVVSSQFFERYFYCLWWG

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156 LQQLSSYGQNLSTTMFMGETTFAVLIAIFGLVLFAHLIGNMQTYLQSLTVRLEEWRLKK RDTEEWMRHRQLPEELRNRVRRYEQYKWL ATRGVDEEVLLQSLPTDLRRDIQRHLCLD LVRRVPFFSQMDDQLLDAICERLVSSLCTEG TYLVREGDLISEMLFIIRGRLESSTTNGGR TGFFNSIILRPGDFCGEELLSWALLPKSTLNLPSSTRTVRALVEVEAFALRAEDLKFVANQ FRRLHSKKLQHTFRFYSHHWRTWAACFIQAAWRRYKRRVMENNLTAIESMENEEGEV GEELVVVEEEECVEESPRTKMNLGVMVLAS RFAANTRRGVAAQRVKDVELPRFKKPEE PDFSAEHDD >atCNG18 MNKIRSLRCLLPETITSASTAASNRGSDGSQ FSVLWRHQILDPDSNIVTYWNHVFLITSIL ALFLDPFYFYVPYVGGPACLSIDISLAATVT FFRTVADIFHLLHIFMKFRTAFVARSSRVF GRGELVMDSREIAMRYLKTDFLIDVAAMLP LPQLVIWLVIPAATNGTANHANSTLALIV LVQYIPRSFIIFPLNQRIIKTTGFIAKTAWAG AAYNLLLYILASHVLGAMWYLSSIGRQFSC WSNVCKKDNALRVLDCLPSFLDCKSLEQPERQYWQNVTQVLSHCDATSSTTNFKFGMF AEAFTTQVATTDFVSKYLYCLWWGLRNLSSY GQNITTSVYLGETLFCI TICIFGLILFTLLI GNMQSSLQSMSVRVEEWRVKRRDTEEWMRHRQLPPELQERVRRFVQYKWLATRGVD EESILHSLPTDLRREIQRHLCLSLVRRVPFF SQMDDQLLDAICGCLVSSLSTAGTYIFREG DPVNEMLFVIRGQIESSTTNGGRSGFFNST TLRPGDFCGEELLTWALMPNSTLNLPSSTRS VRALSEVEAFALSAEDLKFVAHQFKRLQSKKLQHAFRYYSHQWRAWGACFVQSAWRR YKRRKLAKELSLHESSGYYYPDETGYNEE DEETREYYYGSDEEGGS MDNTNLGATILAS KFAANTRRGTNQKASSSSTGKKDGSSTSLKMPQLFKPDEPDFSIDKEDV >atCNG19 MAHTRTFTSRNRSVSLSNPSFSIDGFDNSTV TLGYTGPLRTQRIRPPLVQMSGPIHSTRRT EPLFSPSPQESPDSSSTVDVPPEDDFVFKNANL LRSGQLGMCNDPYCTTCPSYYNRQAAQ LHTSRVSASRFRTVLYGDARGWAKRFASSV RRCLPGIMNPHSKFVQVWTRVLAFSSLV AIFIDPLFFFLLLIQQDNKCIAIDWRATKVLV SLRSITDLIFFINILLQFRLAYVAPESRIVGA GQLVDHPRKIARHYFRGKFLLDMFIVFPIPQIM ILRIIPLHLGTRREESEKQILRATVLFQYI PKLYRLLPLLAGQTSTGFIFE SAWANFVINLLTFMLAGHA VGSCWYLSALQRVKKCML NAWNISADERRNLIDCARGSYASKSQRDL WRDNASVNACFQENGYTYGIYLKAVNLTN ESSFFTRFSYSLYWGFQQISTLAGNLSPSYS VGEVFFTMGIIGLGLLLFARLIGNMHNFLQ SLDRRRMEMMLRKRDVEQWMSHRRLPEDIRKRVREVERYTWAATRGVNEELLFENMP DDLQRDIRRHLFKFLKKVRIFSLMDESVLDSIRERLKQRTYIRSSTVLHHRGLVEKMVFI VRGEMESIGEDGSVLPLSEGDVCGEELLTW CLSSINPDGTRIKMPPKGLVSNRNVRCVT NVEAFSLSVADLEDVTSLFSRFLRSHRVQGAIRYESPYWRLRAAMQIQVAWRYRKRQL QRLNTAHSNSNR >atCNG20 MASHNENDDIPMLPISDPSSRTRARAFTSR SRSVSLSNPTSSIEGFDTSTVVLGYTGPLRT QRRPPLVQMSGPLTSTRKHEPLFLPHPSSD SVGVSSQPERYPSFAALEHKNSSEDEFVLK HANLLRSGQLGMCNDPYCTTCPSYYNRKAAQI PTSRVSALFDSTFHNALYDDAKGWAR RFASSVNRYLPGIMNPHAKEVQTWTKFFALSCLLAIFIDPLFFFLIKVQEQNKCIMIDWP MTKAFVAVRSVTDVIFTMNILLQFRLAYVA RESTVVGAGQLVSHPKKIALHYLKGKFFL DLFIVMPLPQILILWIIPAHLGASGANYAKNLLR AAVLFQYIPKLYRLLPFLAGQTPTGFIF ESAWANFVINLLTFMLAGHVVGSCWYLFGLQ RVNQCLRNACGNFGRECQDLIDCGNG NSSVLVRATWKDNASANACFQEDGFPYGI YLKAVNLTNHSNLFTRYSYSLFWGFQQIST LAGNQVPSYFLGEVFFTMGIIGLGLLLF ALLIGNMQNFLQALGKR NLEMTLRRRDVEQW MSHRRLPDGIRRRV REAERFNWAATRGVNEELLFENMP DDLQRDIRRHLFKFLKKVRIF SLMDEPILDAIRERLKQRTYIGSSTVLHRGGLVEKMVFIVRGEMESIGEDGSVLPLYEGD

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157 VCGEELLTWCLERSSVNPDGTRIRMPSKGLL SSRNVRCVTNVEAFSLSVADLEDVTSLFS RFLRSHRVQGAIRYDSPYWRLRAARQ IQVAWRYRRRRLHRLCTPQSSYSL >ntCNG MTILNQEKYIRFEDCKSEDNRLFSGRKPSTRS WMSSIRRGFSDRLSSLKRKSRCIPSLSDW PKQVSEGSSRNKILDPQEPFLQFWNKIFVLACIVSVAIDPLFFYISVVDIKRKCLDLDHSLK IPISVLRSATDLFYIYHIFGQFRTGFIAPSSRVF GRGELIEDSSLIAKRYIPYCIIDVLAVLPLP QLVLYINAPNANRAISLVMKKQLVIVVFT QYVPRIFRIFPLYREVTRTTGFFTETAWAGA AFNLFLFMIASNVVGALWYLITVERQDNCWSQVCKGFEECVLDHLCCGQQGKNAQFLN FSCRLLKPEEIQENDFDFGIFRDALQSRVVQRRNFWSKLSYCFWWGLRNLSSLGQGLNT SDFLGEILFAVFICILGLILFSLLIGNMQEYLQSITVRVEGMRLRRRDAEQWMSHRMLPD NLRERIRRYEQYKWQQTRGVDEDYLICNLPKDLRRDVKRHLCWSLLKRVPMFEKMDE QLLDALCDRLKPALFTENSFIIREGDPVNE MLFLMRGTLLTITTNGGRTGFFNSASLSAG DFCGEELLTWALDPNASSCLPASTRTVQAVIDVEAFALTADDLKFVAAQFRRLHSKQIR HTFRFYSQHWRTWAACFIQAAWRRHYRNK LEKSLREEEDRLQAALENETANIPSLGATI YASRFAANALRILRRNHPKGS KSSSKVSPLLLQKPAEPDFSS >ptK+ MMMIQGRERTRGGVGNGGDGSD EEEELEVEKLRGESKPSWKRLFGLLIMESPIRDGIVF RDGSGLGQSSVSDAYIIRPDSW RYTVWVHFILIWAVYSSFFTPLEFGFFRGLPENLFLLDI AGQIAFLIDIVVHFFVAYRATHSYRLVCRHKLI AIRYLKSRFLVDFLGCLPWDAIFKVSGR KEAVRYMLWIRLSRAKRVSEFFERLEKDIRI NYLFTRIVKLLVVELYCTHTAACIFYYLA TTMPPSQEGYTWIGSLQMGDYHYTHFREIDLWKRYITSLYFAIVTMATVGYGEIHAVNV REMIFVMVYVSFDMILGAYLLGNMTALIV KGSKTEKFRDRMTDLIKYMNRNNLGKGIS NEIKRHLRLQYDRSYTEASALQ EIPASIRTKISQKLYEPYIKEVSLFKGCSLGFIKQIAIRVH EEFFLPGEVIIEQGQVADQLYVVCHGELEEFGRGENDRAEESTKLLQTYSSFGEVSFLCN TPQPYTIRVRELCRVLRLD KQSFTEILEIYFSDGRIILNN LLEGKDANLRNELLESDVTLYI EKSESELAMRLNCAAFDGDYYRLRQLIE AGADPNKADYDRRSPLHVAASKGDVDISLL LIETWEWTSNISDKFGNTPLLEAVKGGHD EVASLLVKAGASLAIDDAGGFLCTIVVKRD LNLLKRVLANGINPNAKNFDYRTPLHIAAS EDLHSIASLLLEAGASVFPKDRWGHTPLDE ARIGGNKDLIKMLEVARASQIVTDDMQRMKCTVFPFHPWDPKEKRREGVVLWVPQTIE ELVKAAMEQLKSSGGYLLSENGGKILDVNMISHDQKLFLVNE >atAKT1 MRGGALLCGQVQDEIEQLSRESSHFSLSTGI LPSLGARSNRRVKLRRFVVSPYDHKYRIW EAFLVVLVVYTAWVSPFEFGFLRKPRPPLSITDNIVNAFFAIDIIMTFFVGYLDKSTYLIVD DRKQIAFKYLRSWFLLDLVSTIPSEAAMRISSQSYGLFNMLRLWRLRRVGALFARLEKD RNFNYFWVRCAKLVCVTLF AVHCAACFYYLIAARNSNP AKTWIGANVANFLEESLWM RYVTSMYWSITTLTTVGYGDLHP VNTKEMIFDIFYMLFNLGL TAYLIGNMTNLVVHGTS RTRNFRDTIQAASNFAHRNHLPPRLQDQML AHLCLKYRTDSEGLQQQETLDALPKAIRS SISHFLFYSLMDKVYLFRGVSNDLLFQLVS EMKAEYFPPKEDVILQNEAPTDFYILVNGT ADLVDVDTGTESIVREVKAGDIIGEIGVLCYRP QLFTVRTKRLCQLLRMNRTTFLNIIQAN VGDGTIIMNNLLQHLKEMNDPVMTNVLLEIEN MLARGKMDLPLNLCFAAIREDDLLLH QLLKRGLDPNESDNNGRTPLHIAASKGTLN CVLLLLEYHADPNCRDAEGSVPLWEAMV EGHEKVVKVLLEHGSTIDAGDV GHFACTAAEQGNLKLLKEI VLHGGDVTRPRATGTSA LHTAVCEENIEMVKYLLEQGADVNKQDMHG WTPRDLAEQQGHEDI KALFREKLHERR VHIETSSSVPILKTGIRFLGRFTSEPNIRPASREVSFRIRETRARRKTNNFDNSLFGILANQS VPKNGLATVDEGRTGNPVRVTISCAEKDDIAGKLVLLPGSFKELLELGSNKFGIVATKV MNKDNNAEIDDVDVIRDGDHLIFATDS

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158 >atSKOR MGGSSGGGVSYRSGGESDVELEDYEVDDFR DGIVESRGNRFNPLTNFLGLDFAGGSGG KFTVINGIRDISRGSIVHPDNR WYKAWTMFILIWALYSSFFTPLEFGFFRGLPENLFILDIA GQIAFLVDIVLTFFVAYRDSRTYRMIYKRSSI ALRYLKSTFIIDLLACMPWDIIYKAAGEK EEVRYLLLIRLYRVHRVILFFHKMEKDIRINY LFTRIVKLIFVELYCTHTAACIFYYLATTL PASQEGYTWIGSLKLGDYSYSKFREIDLWTRYTTSMYFAVVTMATVGYGDIHAVNMRE MIFAMVYISFDMILGAYLIGNMTALIVKGS KTERFRDKMADIMRYMNRNKLGRNIRGQI TGHLRLQYESSYTEAAVLQDIPVSIRAKIAQTLY LPYIEKVPLFRGCSSEFINQIVIRLHEEF FLPGEVIMEQGSVVDQLYFVCHGVLEEIGIT KDGSEEIVAVLQPDHSFGEISILCNIPQPYT VRVAELCRILRLDKQSFMNILEIFFHDGRRIL NNLLEGKESNVRIKQLESDITFHISKQEAE LALKLNSAAFYGDLYQLKSLIRAGGDPNKTDYDGRSPLHLAASRGYEDITLYLIQESVD VNIKDKLGSTPLLEAIKNGNDRVAALLVKE GATLNIENAGTFLCTVVAKGDSDFLKRLL SNGIDPNSKDYDHRTPLHVAASEGFYVLAI QLVEASANVLAKDRWGNTPLDEALGCGN KMLIKLLEDAKNSQISSFPSGSKEPKDKVYK KKCTVYFSHPGDSKEKRRRGIVLWVPRSI EELIRTAKEQLNVPEASCVLSEDE AKIIDVDLISDGQKLYLAVET >ntNKT2 MIIEDSQIKDQHVQDNNHGS NNSSGTNSDELGFRNLSKL ILPPLGSNDYNQNQTQQKGKI ITPMDSRYRCWETLMVVMV AYSAWVCPFEIAFMRSNPN RALYFADNVVDLFFAVDIIL TFFVAYIDTTTQLLVRGRRRIATRYTSTWF MMDVASTVPFDLLALIFTGKHQIGISYSVL GMLRFWRLRRVKQFFTRLEKDMRFSYFWVR CARLLFVTLLTVHCAGCLYYLLADRYP HQGDTWLGAMNPNYK ETSLLIRYIAALYWSITTMTTVGYGDLHAVNTLEMVFIIFYMLF NLGLTAYIIGNMTNLVVEGTRRTMEFRNSIEAASNFVCRNRLPPRLKEQILAYMCLRFRA ESLNQQQLIEQLPKTICKSIRHHLFLPTVE KVYLFKGVSREILLLLV ADMKAEYIPPREDVI MQNESPDEVYIIVSGEVEMIECEMENEQ VVWTFKSGDMLGEVGAFCCRPQSYTYRTKT LSQLLKIRATSLIEAMKTRQEDNIIMIKNF LQHHKKLRDLKLGDLFHEVGAENGDPNMS VNLLTVASTGNATFLEELLKAR LDPDIGDAQGRTPLHIAASKGHEECVMVLLRHGCNIH LRDVNGNTALWEAIAEKQHPTFRILYHW ASVSDPYVAGELLCTAAKRNDLTVMKELLK HGLIVDSKDRHGSTAIHVALEENHEDMVK LLLMNGAEINDKFKHKLSSMNLSEMLQKR EVGHRVIVSDTMDEVAQKWREQEQKYNS GNTRDQSSFRVSIYKGHPVIRKRTHCSEPG KLIILPNSLAELKIIAGQKFGFDATNALATDQEGSEIDSIEVIRDNDKLFIVEDPKCL >atAKT2 MDLKYSASHCNLSSDMKLRRF HQHRGKGREEEYDASSLSLN NLSKLILPPLGVASYNQN HIRSSGWIISPMDSRYRC WEFYMVLLVAYSAWVYPFEVAFLNSSPKRNLCIADNIVDLFF AVDIVLTFFVAYIDERTQLLVREPKQIAVRYLS TWFLMDVASTIPFDAIGYLITGTSTLNIT CNLLGLLRFWRLRRVKHLFTRLEKDIRYSYFWIRCFRLLSVTLFLVHCAGCSYYLIADRY PHQGKTWTDAIPNFTETSLSIRYIAAIYWSI TTMTTVGYGDLHASNTIEMVFITVYMLFNL GLTAYLIGNMTNLVVEGTRRTMEFRNSIEA ASNFVNRNRLPPRLKDQILAYMCLRFKAE SLNQQHLIDQLPKSIYKSICQHLFLPSVEK VYLFKGVSREILLLLV SKMKAEYIPPREDVI MQNEAPDDVYIIVSGEVEIIDSEMERESVLGTLRCGDIFGEVGALCCRPQSYTFQTKSLSQ LLRLKTSFLIETMQIKQQDNATMLKNFLQH HKKLSNLDIGDLKAQQNGENTDVVPPNIA SNLIAVVTTGNAALLDELLKAKLSPDITDS KGKTPLHVAASRGYEDCVLVLLKHGCNIHI RDVNGNSALWEAIISKHYEIFRILYHFAAI SDPHIAGDLLCEAAKQNNVEVMKALLKQGL NVDTEDHHGVTALQVAMAEDQMDMVNLLA TNGADVVCVNTHNEFTPLEKLRVVEEE EEEERGRVSIYRGHPLERRERSCNEAGKLILL PPSLDDLKKIAGEKFGFDGSETMVTNED GAEIDSIEVIRDNDKLYFVVNKII >zmZMK2

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159 MKKNNSSIESTGGGVGGSGGTVSGSGSGSFN LRNLSKVILPPLGGPSGGQSQSHGGSDK WVVSPLDSRYRWWDTFMVVLVAYSAWV YPFEVAFMNASPKGGLEVADIVVDLFFAV DIVLTFFVAYIDGRTQLLVRDRKKITLRYLST FFIMDVASTIPFQGLAYLITGEVRENAVY SMLGVLRLWRLRRVKQFFTRLEKDIRFSYFWIRSARLVAVTLFLVHCAGCLYYLIADRY PDRQKTWIGAVIPNFRQASLRIRYISSIYW SITTMTTVGYGDLHAQNNVEMIFNIFYMLF NLGLTAYLIGNMTNLVVEGTRRTMEFRNSI RAASSFVGRNHLPPRLKQQILAYMCLKFR AESLNQQQLMDQLPKSICKSICEHLFVPV VKDVYLFRGVSREMLLSLVTKMKPEYIPPKE DVIVQNEAPDDVYVVVSGEVEVILFDGIYE QVQATLGARDIFGEVSALSDRAQAFTFRTR TLSQLLRLKQATLKEAMQSRPEDSVVVIKNFLKHQVEMHGMKVEDLLGDNTGEHDDD AIVLTVAAMGNSGLLEDLLRAGKAADVGDAK GRTALHIAASKGYEDCVLVLLKHACN VNIRDAQGNTAMWNAIAAGH HKIFNLLYQFGRASNPRAGGDVMCLAARRGHLGALQE LLKLGLDVDSEDHDGATALRVAMAEGHA DAARFLILNGASVDKASLDDDGSGSGSGS GAARLAMSPTELRELLQKRELGHSITIHDSP AVVPNGGSSGHSRPGRLQSTSSDSQRWPR VSVYKGHPFLRNRTSEAGKLINLPGTMEEF KVIVGEKLKVDAEKALIVSDEGAEIDSIDVI RDNDKLFMVTEEDLRRLASMDWLSCE >dcK+ MAAETRSPVPLLYRRRSSGEIMRNMASVSSSLLPAFGTVVGDGSPLLRSYIIAPYDRRYR WWQAFLVILVIYSAWSSPFELAFKDVATGS LLPVDLVVDAFFAIDIILTFFVAYLDKSTYL LVDDHKQIAVRYVTHLWFPMDLASTLPSQTI YRIFAGEMHHGEVFGFLNLLRLWRLRRV SELFSRLEKDTRFSYFWTRYCKLIAVTLFAVHSAACFYFWLAIHHKIPEQTWIGAQVDNF ENRSIWLGYTYAMYWSIVTLTTTGYGDLYSKNTGEKVFNIFYMLFNIGLTAYLIGNMTN LIVHSAIKTFAMRDAINEVLRYASKNRLPEGLKEQMLAHMQLKFKTAELQQEEVLEDLP KAIRSSIAQHLFHKTIENTYLFRDVSDDLISQLVSEIKAEYFPPKVEIILQNEIPTDFYVIAS GAVDVVTQKNGIEQFVTKLSSKEMFGDIGVIFN IPQPFTVRTRRLSQVIRISHHSFKDMM QPHNEDGQKILRNFILYLKGLQKEVLDEIP FLSDMLGDLNNEHSGLLDQSQEIEPSNYDQ GENAQGSHVDSAFQSAYPIRLVIHGHHPDL ETEDKGTGKLIHLPESMEGLLMLAEKKFG KKGDIVLMEDGSQVEDLDALRENDHLYIF >osAKT1 MPTTKCAVPLVSGAAGGGGS AELTRQLSSTQASPRFSFSSG VLPSLGSRGGGERHARLR RFIVSPYDRRYELWNNYLILLVVYSAWVTP FEFGFVPEPAGALAAADNAVNAFFAVDIV LTFFVAYTDPKTFLLQDDPRKIA LRYITTWFVLDVVATIPTELA RRILPPDLRSYGFFGILR LWRLHRVGILFARLEKDRKFSYFWVRCVKLVCVTLFAVHCSACFYYLLADRYPDPTNT WISAYMPNFHKASIWSRYVASMYWSITTL STVGYGDMHAENTGEMVFTTTYMLFNLG LTAYIIGNMTNLVVHGTSRTRK FRDMIQAATSFAQRHQLPAR LQEQMVSHLSLKFRTNS EGLHQQETFEALPKAIKSSISHHLFFGLVQNVYL FEGVSNDLIFQLVSEMNAEYFAPREDI ILQNEAPADFYIIVSGSMELIELHNGIEQASV LTLAGMAKSGDVVGEIGVLCYRPQLFTAR TRSLCQLLRLDRAAFLRIIQSNIADGTIVMNNL IQYLREKKEIASIVAVAKEIDDMLARGQ MDFPITLCFAASKGDSFLLHQLLKRGLD PNESDHYGRTALHIA ASNGNEQCVRLLLENG ADSNSRDPEGRVPLWEALCRRHQTVVQLLVDAGADLSGGDAAPYARVAVEQNDAALL GEIVRHGGDVSGACSGDGTTALHRAVL DGNVQMARLLLEHGADADAEDVNGLTPRAV AEQGGHADMQLAFASATRHEPRKARPPPP ASAIVPVPLRDGVDSSPSSSSRRGRTSSTSA ASARSTPQRMANFRNSLFGVISSSHAFHHE GGYRGGGGGGGAAAER ERSSSSPPLVRVA ISCPESRGGKDHSSKLVFMPETLRGLLEL GAARFGVSPTRVVTSGGADVDDARLVRDGD HLLLVTDKWVPPENRSRNQ >rnEAG7

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160 MPVRRGHVAPQNTFLGTIIRKFEGQNKKFIIANARVQNCAIIYCNDGFCEMTGFSRPDVM QKPCTCDFLHGPETKRHDIAQIAQALLGSEER KVEVTYYHKNGSTFICNTHIIPVKNQEG VAMMFIINFEYVTDEDNAASPERVNPILPVKSVNRKLFGFKFPGLRVLTYRKQSLPQEDP DVVVIDSSKHSDDSVAMKHFKSPTKESCSP SEADDTKALIQPSQ CSPLVNISGPLDHSSPK RQWDRLYPDMLQSSSQLTHSRSRESLCSIRR ASSVHDIEGFNVHPKNIFRDRHASEDNGR NVKGPFNHIKSSLLGSTSDSNLNKYSTINKIPQLTLNFSDVKTEKKNTSPPSSDKTIIAPKV KERTHNVTEKVTQVLSLGADVL PEYKLQTPRINKFTILHYSPFKAVWDWLILLLVIYTAI FTPYSAAFLLNDREEQKRRECGYSCSPLNVVDLIVDIMFIIDILINFRTTYVNQNEEVVSD PAKIAVHYFKGWFLIDMVAAIPFDLLIFGSGSDETTTLIGLLKTARLLRLVRVARKLDRY SEYGAAVLMLLMCIFALIAHWLACIWYAI GNVERPYLTDKIGWLDSLGTQIGKRYNDSD SSSGPSIKDKYVTALYFTFSSLTSVGFGNVSPN TNSEKIFSICVMLIGSLMYASIFGNVSAII QRLYSGTARYHMQMLRVKEFIRFHQIPNPL RQRLEEYFQHAWTYTNGIDMNMVLKGFP ECLQADICLHLNQTLLQNCKAFRGASKGCL RALAMKFKTTHAPPGDTLVHCGDVLTAL YFLSRGSIEILKDDIVVAILGKNDIFGEMV HLYAKPGKSNADVRALTYCDLHKIQREDLL EVLDMYPEFSDHFLTNLELTFNL RHESAKSQSINDSEGDTCK LRRRRLSFESEGDKDFSK ENSANDADDSTDTIRRYQSSKKHFEEKKS RSSSFISSIDDEQKPLFLGTVDSTPRMVKASR HHGEEAAPPSGRIHTDKRSHSCKDITDTHSWEREHARAQPEECSPSGLQRAAWGISETES DLTYGEVEQRLDLLQEQLNRLESQMTTDIQAILQLLQKQTTVVPPAYSMVTAGAEYQRP ILRLLRTSHPRASIKTDRSFSPSSQCPEFLDL EKSKLKSKESLSSGKR LNTASEDNLTSLLK QDSDASSELDPRQRKSYLHPIRHPSLPDSSLSTVGILGLHRHVSDPGLPGK >hsHERG MPVRRGHVAPQNTFLDTIIRKFEGQSRKFIIANARVENCAVIYCNDGFCELCGYSRAEVM QRPCTCDFLHGPRTQRRAAAQIAQALLGAEERKVEIAFYRKDGSCFLCLVDVVPVKNED GAVIMFILNFEVVMEKDMVGSPAHDTNHRGPPTSWLAPGRAKTFRLKLPALLALTARES SVRSGGAGGAGAPGAVVVD VDLTPAAPSSESLALDEVTAMDNHVAGLGPAEERRALV GPGSPPRSAPGQLPSPRAHSLNPDASGSSCSLARTRSRESCASVRRASSADDIEAMRAGV LPPPPRHASTGAMHPLRSGLLNSTSDSDLVRYR TISKIPQITLNFVDLKGDPFLASPTSDRE IIAPKIKERTHNVTEKVTQVLSLGADVLPE YKLQAPRIHRWTILHYSPFKAVWDWLILLL VIYTAVFTPYSAAFLLKETEE GPPATECGYACQPLAVVDLIVDI MFIVDILINFRTTYVNA NEEVVSHPGRIAVHYFKGWFLIDMVAAIPFD LLIFGSGSEELIGLLKTARLLRLVRVARK LDRYSEYGAAVLFLLMCTFALIAHWLACIW YAIGNMEQPHMDSRIGWLHNLGDQIGKP YNSSGLGGPSIKDKYVTALYFTFSSLTSVGF GNVSPNTNSEKIFSICVMLIGSLMYASIFG NVSAIIQRLYSGTARYHTQMLRVREFIRFH QIPNPLRQRLEEYFQHAWSYTNGIDMNAV LKGFPECLQADICLHLNRSLLQHCKPFRGATKGCLRALAMKFKTTHAPPGDTLVHAGD LLTALYFISRGSIEILRGDVVVAILGKNDIFG EPLNLYARPGKSNGDVRALTYCDLHKIHR DDLLEVLDMYPEFSDHFWSSL EITFNLRDTNMIPGSPGS TELEGGFSRQRKRKLSFRRRT DKDTEQPGEVSALGPGRAGAGPSSRGRPGGPWGESPSSGPSSPESSEDEGPGRSSSPLRL VPFSSPRPPGEPPGGEPLMEDCEKSSDTCNP LSGAFSGVSNIFSFW GDSRGRQYQELPRCP APTPSLLNIPLSSPGRRPRGDVESRLDALQRQLNRLETRLSADMATVLQLLQRQMTLVPP AYSAVTTPGPGPTSTSPLLPVSPLPTLTLDSLS QVSQFMACEELPPGAPELPQEGPTRRLSL PGQLGALTSQPLHRHGSDPGS >dmERG MSHKSCVELSEIRKLDKIVQQCELYMSNNNI NDNAAKKPPVKEFKRNCPAKSNKFVERE LFSLMCNLNKIEKKQVFVSDILINASMFD DKKESLARFPKNVSDHNTLVYHKFLRTRPTN DDEALKLKAGGKSKERPGTKITADCFFSPL LFKINNKRECLKKVK PMKGENAVETNGIP LESKPSQHKKQKSFKKCIKCDKKKLYIKN YQVSARRECDLLCSYDPLRKNNADLFQTHK

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161 GTSYECMFKNLKYNQAEKPTDNVMGNSYDDM LDKEALLGSKSEPKGQDPNDMITSLG GNILLDQKLQNNYYHKWTLLHYSPFKAVWDW IILILVMYTAIFTPYVAAFLLGEQDYQR RNSKYINSDPIVIIDLIVDVT FIVDIIINFRTTFVNSQDEVVSH PGRIAVHYLSGWFLIDLVA AVPFDLLLVGSDTDETTTLIGLLKTARLLRLVRV ARKIDRYSEYGAAVLILLMATFILIAH WLACIWYAIGNAEKSIASKNIGWLNSLAYDIQEPYFDNRTGGPSIKSRYITALYFTFTSLT SVGFGNVAPNTDAEKAFTICVMLVGSLMYASIFGNVSAIIQRLYSGTARYHTQMLRVRE FIRFHQIPNPLRQRLEEYFQHAWTYTNGIDM NSLLKGFPECLQADICLHLNRKLLTTCAA FSEASPGCLRAFSLKFKTTHAPPGDILVHRGDVLTSLYFIARGSIEIQRAGNIVVLGKNDIF GENPCIYPTVGKSNGVVRALTYCDIHKL HRDDLLDVLDSYPEFLESFVSNLVITYNMRD DEHSGVDIKHRYLRAKSSDKM RSSPDIPSIRIVGLRYKKQN VNTSVHKVKNDNSRDLNIF IENEIANYHLDLFDNNN >ceERG MSSSTNHTGIIQHHPSQSQQQATTSSGAGNAV ASQAKQLMVVLQSGSYKVLSLGADVL PEYKLQPTRIHHCTIVHYSPFKAVWDWIILLLV IYTAVFTPYVAAFLLRELQDTAKKSRFT EPLEIVDLIVDIMFIVDIIINFR TTYVNENDEACQVVSDPGKIATHYFKGWFIIDMVAAVPF DLLLVSTNSDETTTLIGLLK TARLLRLVRVARKLDRYS EYGAAVLLLLMATFALIAHWL ACIWYAIGSAELSHKEYTWLHQLSKQLAQPY TSTNGTIPTGGPTLKSR YVTSLYFTLSTIT SIGFGNVSATTDSEKIFTIIMMILGSLMYASVFGNVSAIIQRLYSGTARYHTEMSRLREFIR FHQIPNPLRQRLEEYFQHAWSYTNGIDMNL VLKGFPDCLQADICLHLNRNLLSGCAAFA GSTPGCLRALSMRFRTTHSPPGDTLVHRGDILTGLYFIARGSVEILNDDNTVMGILGKDD IFGENPLLYDEVGKSSCNVRALTYCDLHKILRDDLLDVLDMYPEFAETFCKNLTITYNLR DDAQSLRKKFDRHKLLRMSSSMNKDRYTTPPD GDHGNAAVRRSAESVSRCDSNPIDRR QSAGSRSSSRCSPPHAALTATRSEATPLLRRSTNHHEEDDALFDDIRAFARGNTVTMSPT VAGNSVSPTTAIHNDGIHSQQLSDRSDDYEERRANMFGRRLESIESQMERMQNKFNSDM ETLIKLVKEQSIIRNNGSSNEEPNARYRPNNY ISSAIRLPNGGGGGV VDEMRVSRLSSHEP PTPTQETDTIL

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162 Figure B-1. Alignment of CNBD-containi ng potassium and cation channels used for construction of the phylogenetic tree. This alignment was obtained using ClustalX followed by the removal of all gaps in GeneDoc. Abbreviations: hs Homo sapiens (human), dm Drosophila melanogaster pa Panulirus argus (lobster), ac Aplysia californica sp Strongylocentrotus purpuratus (sea urchin), rn Rattus norvegicus (rat), ce Caenorhabditis elegans at Arabidopsis thaliana os Oryza sativa (rice), rp Rhodopseudomonas palustris bj Bradyrhizobium japonicum mm Magnetospirillum magnetotacticum pt Populus trichocarpa (aspen), dc Daucus carota (carrot), nt Nicotiana tabacum (tobacco), zm Zea mays (corn), lp Limulus polyphemus (horseshoe crab), ml Mesorhizobium loti te Trichodesmium erythraeum pa Paramecium aurelia

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163 APPENDIX C RECORDING OF THE PHYSALIA VOL TAGE-GATED POTASSIUM CHANNEL EXPRESSED IN OOCYTES Figure C-1. Recording of the Physalia voltage-gated potassium cha nnel expressed in oocytes. In response to depolarization from the holdi ng potential of -80 mV to +50 mV (in +10 mV steps) an instan taneous outward current which decreased to a smaller steadystate current was observed. This is the characteristic current expected of the Physalia voltage-gated potassium channel.

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164 APPENDIX D CLONING AND LOCALIZATION OF A CYCL IC NUCLEOTIDE-GATED CHANNEL IN APLYSIA CALIFORNICA CNG Cloning and Probe Synthesis Analysis of the A. californica transcriptome and preliminary genomic data showed the existence of six fragments corresponding to subunits of a CNG channel. Comparison with the CNG channels of other organisms showed that th ese fragments would overlap if they belonged to the same transcript, but assembly of these fragments in SeqMan (DNASTAR) did not produce any alignment. This suggests that potentially up to six subunits and one or two -subunits of the CNG channel exist in A. californica However, most of these fragments have a low E-value (e-6 or less). Primers (5'-CTACAGACCTC TTTTACTTCCTCAAC-3' and 5'-CTTGATCACCCTTTCCTCGAGCTC-3') were designed to the on ly CNG fragment with a good E-value (e-66). A 545 nucleotide frag ment was obtained by PCR from the whole CNS library. This fragment was then ligate d into pCR4-TOPO vect or (Invitrogen), and transformed into One-Shot competent E. coli cells (Invitrogen). The clones were isolated, purified using a Qiagen Kit and sequenced by the Whitney Laboratory molecular core facility. A probe for in situ hybridization corresponding to the w hole obtained fragment was then synthesized. Localization of the CNG RNA in the CNS of A. californica Similar to acHCN, the cloned CNG channel transcript expressed most abundantly in presumable motoneurons, specifically in 11-14 neur ons (90-120 m in diameter) in the A cluster of the cerebral ganglion (Figure D-1, n=3). Al so, six-nine neurons (15-45 m in diameter) stained between cerebropleural and pleuroabdomi nal connectives of left and right pleural ganglia. The transcript did not express in sensory cells or serotonergic neurons.

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165 There was only very weak staining in pedal ganglia, a model to st udy nerve injury in A. californica Because the CNG transcript expressed cons istently only in the cerebral ganglion, I did not determine how expression of this transc ript changes following ne rve injury, because the cerebral ganglion in A. californica is unpaired, thus there is no control for this study. Discussion Similarly to HCN, CNG channel is also activated by cyclic nucleotides. In vertebrates, CNG channels are most strongly ex pressed in sensory cells, such as rod and cone photoreceptors (Yau and Baylor, 1989), extraret inal photoreceptors of pineal gland (Dryer and Henderson, 1991) and parietal eye (Finn et al., 1997) and olfactory neurons (Frings et al., 1995) where they participate in signal transduction. In inverteb rates, CNG channels were found exclusively in sensory organs and associated neuron al structures: eyes and antennae of D. melanogaster (Baumann et al., 1994) and several of its neuronal regions responsib le for processing of visual and olfactory information, incl uding the medulla, lobulla and l obulla plate, the antennal lobe glomeruli, and mushroom bodies (Miyazu et al ., 2000); olfactory, gustato ry, and thermosensory neurons of C. elegans (Coburn and Bargmann, 1996); hype rpolarizing and depolarizing photoreceptors of some molluskan species (McReynolds and Gorman, 1974, Johnson et al., 1986); and (4)ventral eye nerves of Limulus polyphemus (Chen et al., 1999). Thus, it came as a surprise to find expression of the cloned CNG channel in the putative motoneurons of the A cluster in the cerebral ganglion and not in the sensory cells. These motoneurons were shown to control pedal and parapodial movements in A. californica (Jahan-Parwar and Fredman, 1978). Therefore, in mollusks, CNG channels might have evolved to perform differe nt functions than in chordates or arthropods, e.g., they may play a role in controlling locomotion. However, the possibility remains that all or some of the other potential A. californica CNG channels do express in sensory neurons. This pos sibility can be tested after more genomic and

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166 transcriptomic data on the rest of the potential CNG channels in A. californica becomes available.

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167 Figure D-1. Expression of an A. californica CNG channel transcript as determined by in situ hybridization. The strongest staining is in the A-cluster neurons of the cerebral ganglion, which control pedal and parapodial movements. There was also staining of several cells in left and right pleural ganglia and weak stai ning in pedal ganglia. A) Cerebral ganglion. MCC-metacerebral cells. B) Left pedal ganglion. C) Left pleural ganglion. D) Right pleural ganglion. E) Abdominal ganglion. F) Buccal ganglia. Scsensory clusters. Scale bars in A, B and E= 500 m; scale bars in C, D and F=200 m

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181 BIOGRAPHICAL SKETCH Pavlo Kuzyk was born in Rostov-on-Don in 1979. He graduated from Experimental School-Laboratory in Ivano-Frankivsk, Ukrain e, in 1996. Biology was his passion since his childhood and already in the middle school Pavl o wrote a manuscript about local birds and successfully defended it earning th e second place in the republican Olympiad. His other passion was drawing and in 1995 he graduated with a di ploma cum laude from the Art School in IvanoFrankivsk, Ukraine. However, he chose biology as his career and following graduation from the High School, he studied in the Lviv National Un iversity, Ukraine, majoring in microbiology. Pavlo graduated in 2001 with a diploma cum laude. He then moved to US and for the next year worked in the laboratory of Dr. Ronald Calabres e at the Emory University Atlanta, studying the neuronal network controlling heartbea t in the leech. In the fall of 2002 he joined Interdisciplinary Program in the Biomedical Sciences at the Univer sity of Florida and proceeded to work with Dr. Leonid Moroz in the Whitney Laboratory for Ma rine Bioscience, Saint Augustine, Florida.