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Molecular cloning and genetic characterization of the mammalian and nematode nca gene family of four… Hamming, Kevin Scott Christopher 2004

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MOLECULAR CLONING AND GENETIC CHARACTERIZATION OF THE MAMMALIAN AND NEMATODE nca GENE FAMILY OF FOUR DOMAIN-TYPE VOLTAGE-GATED ION CHANNELS by KEVIN SCOTT CHRISTOPHER HAMMING B.Sc, Simon Fraser University, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Graduate Program in Neuroscience) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 2004 © Kevin Hamming, 2004 Abstract Voltage-gated ion channels (VGICs) are involved in numerous physiological processes including cellular excitability, electrical signaling and neurotransmitter release. VGICs have also been implicated in human diseases such as night blindness, migraine headaches, cardiovascular disease and certain movement and muscle disorders. While significant work has been performed towards identifying the different types of VGICs in native cells and their molecular counterparts, there still are a number of native conductances that remain to be fully characterized. The role that these "new" channels may play in normal physiological processes and disease states is not known. Thus, a complete study of these new channels is necessary to understand their basic properties and contributions to neuronal physiology. The goals of this study were to identify novel four domain-type VGICs and determine their physiological functions in mammalian and nematode model systems. Screening of the C. elegans genome and GenBank EST databases identified the nca family of four domain-type VGICs. Sequence comparisons between representative Ca 2 + and Na + channel ot(i) subunits and that of the NCA channels revealed that the NCA channels form their own distinct family of VGICs based upon amino acid identity. Furthermore, these comparisons predict that the NCA channels may have unique ion selectivity, activation and inactivation properties. Despite repeated attempts under a variety of assay conditions, no functional currents were obtained for the full-length NCA channels transiently transfected into HEK tsA201 cells. The physiological functions of the nca-1 and nca-2 genes in C. elegans were examined using deletion mutant strains that contained predicted null mutations. The cellular expression patterns of nca-1 and nca-2 were determined using promoter: :GFP reporter constructs. Both genes were found to be coexpressed in the cholinergic motor neurons of the dorsal and ventral nerve cords and in the GABAergic neuron DVB. The behavioral defects in various mutant ii animals were analyzed using locomotion and defecation assays and revealed that nca-1 and nca 2 may function redundantly in some neurons regulating these two behaviors. A model is proposed that describes the potential roles of the NCA-1 and NCA-2 channels in electrical signaling within the cholinergic motor neurons of the dorsal and ventral nerve cords. iii Table of Contents ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES xii LIST OF TABLES xv LIST OF ABBREVIATIONS xvi ACKNOWLEDGEMENTS xxi DEDICATION xxii CHAPTER 1. INTRODUCTION 1 Evolution of Four Domain-Type Voltage-Gated Ion Channels 2 Four Domain-Type Voltage-Gated Ion Channels 6 Calcium Channels 6 Sodium Channels 15 Ion Channelopathies of Four Domain-Type Voltage-Gated Ion Channels 21 Calcium Channelopathies 22 aisSubunit 22 iv CCIFSubunit 23 aw Subunit 23 Accessory Subunits 26 Sodium Channelopathies 26 Caenorhabditis elegans as a Genetic Model for the Study of Voltage-Gated Ion Channels 27 Voltage-Gated Ion Channel Genes in C. elegans 30 Calcium Channels 30 unc-2 30 egl-19 31 unc-36 32 cca-1 33 Additional Calcium Channel Genes 33 Sodium Channels 35 Potassium Channels 35 Research Objectives: Molecular Cloning and Genetic Characterization of Novel Four Domain-Type Voltage-Gated Ion Channels 35 CHAPTER 2. MATERIALS AND METHODS 37 Screening of Genome Databases for Novel Voltage-Gated Ion Channels 37 Identification of the nca-1, nca-2 and rat-nca Genes 37 Genetic Analyses 39 Nematode Strains and Growth Conditions 39 Isolation of VC12 nca-1 (gk9)IV md VC9 nca-2(gk5)III Deletion Mutant Strains 40 Construction of the TS65 nca-2 (gk5)III; nca-1 (gk9)IV Double Mutant Strain 44 v Construction of cca-1; nca Mutant Strains 45 Construction of unc-2; nca Mutant Strains 45 Construction of unc-2(0x8) cca-1 (adl650); nca Mutant Strains 46 Construction Of unc-25; nca Mutant Strains -46 Construction of the TS199 valsll; valslS Transgenic Strain 47 DNA Transformation and Microinjection of C. elesans 48 Identification of Transformants and Establishing Transgenic Lines 49 Integration of Extrachromosomal Arrays 49 Fluorescent Microscopy of C. elegans 50 Phenotvpic Analysis of nca Mutants 51 Aldicarb and Nicotine Resistance 51 Halothane Assay 52 Body Bends Assay 53 Thrashing Assay 54 Defecation Assay 54 Molecular Biology 55 Isolation of C. elegans Genomic DNA for Genotype Testing 55 RNA Extraction from C. elegans 58 RNA Extraction from Rat Tissues 59 PCR Amplification Procedures 59 RT-PCR 59 PCR 60 5' RACE 61 Site-Directed Mutagenesis 62 Southern Blotting 63 vi Colony Blots 63 Generation of Radioactive Probes and Membrane Hybridization 64 Screening of a ^ ZAPII Rat Brain cDNA Library 65 Constructs Generated for This Study 67 cDNA Clones 67 nca-1 67 The Full-Length "nca-1" cDNA 68 The Full-Length "nca-1 AS" cDNA 71 nca-2 71 The Full-Length "nca-2 " cDNA 72 The Full-Length "A5' nca-2 " cDNA 75 xdX-nca 75 The Full-Length "rat-ncaA, F10-9" cDNA 76 The Full-Length "rat-nca B, FL Ul" cDNA 80 rat-ncaA:.FLAG, "F10-9:.FLAG" 83 Promoter::GFP Fusion Constructs 84 pnca-1 ::G¥P 84 pnca-2::GFP 87 nca-1 Promoter: :DsRed2 Fusion Construct 88 Identity and Similarity Analysis of Four Domain-Type VGIC Subunits 89 In vitro Translation 90 Protein Isolation from Rat Brain and Human Embryonic Kidney tsA201 Cells 90 Generation of Antibodies and Immunohistochemistry 91 Fusion Protein Construction 91 Fusion Protein Expression, Purification and Analysis 92 vii Generation of Polyclonal Antisera 93 Western Blotting 93 Immunofluorescent Staining 94 Electrophysiological Recordings from HEK tsA201 Cells 95 CHAPTER 3. MOLECULAR CLONING AND EXPRESSION OF THE NEMATODE AND MAMMALIAN nca GENE FAMILY 98 Background 98 Results 99 Identification of the nca-1, nca-2 and xzX-nca Genes 99 The Structure of the nca-1 Gene 100 Comparison of the Gene Structure of nca-1 with that Predicted in WormBase 103 The Predicted NCA-1 Protein Products 105 The Structure of the nca-2 Gene 105 Comparison of the Gene Structure of nca-2 with that Predicted in WormBase 112 The Predicted NCA-2 Protein Products 113 Cloning and Sequencing of rat-nca 117 Sequence Analysis of Rat-nca A 118 Comparison of the Primary Structure of NCA-1, NCA-2 and rat-NCA 122 NCA-1/NCA-2 122 NCA-l/rat-NCA 124 NCA-2/rat-NCA 130 Comparison of NCA-1, NCA-2 and rat-NCA with Other VGIC a 0 ) Subunits 130 S4 Regions 134 viii P-Loop Regions 137 rzA-nca Regional RNA Expression 138 Electrophysiological Analysis of HEK tsA201 Cells Tranfected with Rat-nca A 144 Rat-NCA Expression in Transfected HEK tsA201 Cells and Rat Brain Homogenates 146 CHAPTER 4. GENETIC AND PHENOTYPIC ANALYSIS OF nca-1 AND nca-2 MUTANT STRAINS 148 Background 148 The Locomotion Circuit in C. elegans 149 The Defecation Motor Program in C. elegans 153 Results 159 Isolation of Deletion Mutations in the nca-1 and nca-2 Genes 159 Molecular Analysis of the nca-1 (gk9) and nca-2(gk5) Deletion Alleles 159 nca-1 Gene Expression Pattern 163 nca-2 Gene Expression Pattern 167 Coexpression of the nca-1 and nca-2 Genes 172 Phenotypic Analysis of nca Mutant Strains 172 Locomotion 172 Body Bends and Thrashing 177 Aldicarb and Nicotine Assays 182 Halothane Assay 187 Interactions Between nca-1, nca-2 and the unc-2 Ca 2 + Channel Gene 188 Interactions Between nca-1, nca-2, cca-1 and unc-2 VGIC Genes 197 Interactions Between nca-1, nca-2 and unc-25 201 ix Analysis of the Defecation Motor Program in nca Mutant Strains 209 Failure Rate of Defecation 209 Cycle Timing 210 CHAPTER 5. DISCUSSION 215 The Nematode and Mammalian nca Gene Family 215 The Genomic Organization of the nca-1 and nca-2 Genes 217 The nca Genes Likely Encode Members of a Novel Family of Voltage-Gated Ion Channels..219 Comparisons Between nca Family Members 219 Comparisons Between the nca Family and Representative Ca2* and Na+ Channels 221 Cytoplasmic Regions 221 S4 Regions 222 P-Loop Regions 224 NCA Channel Expression and Electrophysiological Analysis 227 Deletion Mutations in the nca-1 and nca-2 Genes 229 The Expression Pattern of the nca Family of Voltage-Gated Ion Channels 231 NCA-1 and NCA-2 are Involved in Acetylcholine Release at the Neuromuscular Junction....234 A Hypothetical Model for the Role of NCA-1 and NCA-2 in Cholinergic Motor Neurons 236 The Effect of Halothane on the nca-2(gk5); nca-1 (gk9) Double Mutant Strain 240 Mutations to both nca-1 and nca-2 Enhance the Locomotion Defects of unc-2 Mutants 242 Possible Calcium Channel Redundancy at the Cholinergic Neuromuscular Junction 245 Mutations at the GABAergic Neuromuscular Junction Partially Suppresses the Locomotion Defect of the nca-2(gk5); nca-1(gk9) Double Mutant 249 Analysis of the Defecation Motor Program in nca Mutant Strains 252 x Failure Rate of Expulsion 252 Cycle Timing 254 Conclusions and Future Studies 255 REFERENCES 262 APPENDIX I. PUBLICATIONS 282 xi List of Figures FIGURE 1. Diagrams Representing the Proposed Evolutionary Events that Have Given Rise to the Four Domain-Type Families of VGICs 4 FIGURE 2. Composition of a HVA Ca 2 + Channel Complex and Structure of the ai Subunit 9 FIGURE 3. Identity Tree Of Mammalian Ca 2 + Channel on Subunits.. 12 FIGURE 4. Composition of a Na+ Channel Complex and Structure of the a Subunit 17 FIGURE 5. Identity Tree Of Rat Na + Channel a Subunits 19 FIGURE 6. Diagrams Illustrating the PCR Strategies Used to Identify Wild-type Versus Deletion Alleles for nca-1, nca-2 and cca-1 41 FIGURE 7. Diagram Illustrating the Cloning Strategy Used to Generate Full-Length nca-1 cDNAs 69 FIGURE 8. Diagram Illustrating the Cloning Strategy Used to Generate Full-Length nca-2 cDNAs 73 FIGURE 9. Diagram Illustrating the Cloning Strategy Used to Generate the Full-Length rat-nca A cDNA 77 FIGURE 10. Diagram Illustrating the Cloning Strategy Used to Generate the Full-Length rat-nca B cDNA 81 FIGURE 11. Diagrams Illustrating the Genomic Region Used to Construct the pnca-l::GFP and pnca-2::G7P Reporter Constructs 85 FIGURE 12. The Structure of the nca-1 Gene and Predicted Protein Products 101 FIGURE 13. The Complete Amino Acid Sequence of the NCA-1 <xi Subunit 106 FIGURE 14. The Structure of the nca-2 Gene and Predicted Protein Products 109 FIGURE 15. The Complete Amino Acid Sequence of the NCA-2 cti Subunit 114 xii FIGURE 16. The Complete Amino Acid Sequence Of The rat-NCA ai Subunit 119 FIGURE 17. Alignment Of The NCA-1, NCA-2 and rat-NCA Amino Acid Sequences 125 FIGURE 18. Identity Tree Of Four Domain-Type VGIC ^ Subunits 132 FIGURE 19. Comparison of S4 Regions Between the NCA, Ca 2 + and Na + Channels 135 FIGURE 20. P-Loop Comparison Between the NCA, Ca 2 + and Na + Channels 139 FIGURE 21. RNA Expression Pattern of rat-nca as Determined by RT-PCR 142 FIGURE 22. The Locomotory Circuit in C. elegans 151 FIGURE 23. The Muscle Contractions of the Defecation Motor Program in C. elegans 155 FIGURE 24. A Model Illustrating the Role of Intracellular Ca 2 + in Regulating the Timing of the Defecation Motor Program in C. elegans : 157 FIGURE 25. Diagrams Illustrating the Molecular Lesions in nca-1 (gk.9) and nca-2(gk5) 161 FIGURE 26. Expression of the nca-1 promoter: :GFP Fusion Construct (p«ca-/::GFP) in Transgenic Animals 164 FIGURE 27. Expression of the nca-2 promoter::GFP Fusion Construct (pnca-2::GFP) in Transgenic Animals 169 FIGURE 28. Coexpression of the nca-1 promoter: :DsRed2 Fusion Construct and the nca-2 promoter::GFP Fusion Construct in the Ventral Nerve Cord 173 FIGURE 29. Coexpression of the nca-1 promoter: :DsRed2 Fusion Construct and the nca-2 promoter::GFP Fusion Construct in the GABAergic Neuron DVB 175 FIGURE 30. Body Bends and Thrashing Analysis of nca Mutant Strains 178 FIGURE 31. Percentage of nca Mutant Animals Paralyzed Over Time on 1 mM Aldicarb 184 FIGURE 32. Halothane Concentration-Response Curves for the nca Mutant Strains 189 FIGURE 33. Thrashing Analysis of unc-2; nca Mutant Strains 192 FIGURE 34. Thrashing Analysis oicca-l(adl650);nca Mutant Strains 198 FIGURE 35. Thrashing Analysis of VGIC Mutant Strains 202 xiii FIGURE 36. Thrashing Analysis of unc-25(el56); nca Mutant Strains 206 FIGURE 37. Defecation Analysis of the nca Mutant Strains 211 FIGURE 38. A Hypothetical Model for the Role of NCA-1 and NCA-2 in Cholinergic Motor Neurons •. 238 xiv List of Tables TABLE 1. Four Domain-Type Voltage-Gate Ion Channels in C. elegans 34 TABLE 2. Oligonucleotide Sequences Used to Screen the C. elegans Genome Database 38 TABLE 3. Sequence of Oligonucleotide Primers 56 TABLE 4. Amino Acid Percent Identity/Similarity Between NCA-1, NCA-2 and rat-NCA 123 TABLE 5. Amino Acid Percent Identity/Similarity Between Four Domain-Type VGIC a ( 1 ) Subunits 131 TABLE 6. Summary of Tentatively Identified Neurons Expressing the pnca-J::GFP Reporter Construct.. : 166 TABLE 7. Summary of Tentatively Identified Neurons Expressing the pnca-2: :GFP Reporter Construct 171 TABLE 8. Summary of the Body Bends and Thrashing Rates for nca Mutant Strains 181 TABLE 9. Summary of the Aldicarb, Nicotine and Halothane Assays 186 TABLE 10. Summary of Thrashing Rates for Different unc-2(ox8); nca Mutant Strains 195 TABLE 11. Summary of Thrashing Rates for Different unc-2(el29); nca Mutant Strains 196 TABLE 12. Summary of Thrashing Rates for Different cca-l(adl650); nca Mutant Strains.... 200 TABLE 13. Summary of Thrashing Rates for Different VGIC Mutant Strains 204 TABLE 14. Summary of Thrashing Rates for Different unc-25(el56); nca Mutant Strains 208 TABLE 15. Summary of the Defecation Results for the nca Mutant Strains 214 xv List of Abbreviations aa amino acid(s) aBoc anterior body muscle contraction ACh acetylcholine AChE acetylcholinesterase AMPA a-amino-3-hyaroxy-5-methyl-4-isoxazolerjropionic acid BDM 2,3-butanedione monoxime bp basep_air(s) BSA bovine serum albumin (Fraction V) BWM body wall muscle Ca 2 + calcium CAN calcium-activated non-selective cation cca calcium channel oti subunit ccb calcium channel £ subunit cDNA complementary deoxyribonucleic acid CGC Caenorhabditis elegans Genetics Center CIN command interneuron CNS central nervous system DABCO l,4-diazabicyclo[2.2.2]octane DEPC diethylpyrocarbonate dH20 distilled water DIC differential interference contrast DHPs 1,4-dihydropyridines DNA deoxyribonucleic acid xvi dNTPs deoxyribonuleoside triphosphates ds double stranded DsRed Discosoma red fluorescent protein DTT dithiothreitol EA-2 episodic ataxia type-2 EC50 concentration of compound that produces 50% of initial response ECL enhanced chemiluminescence EDTA disodium ethylenediaminetetraacetate EGTA ethylene glycol-bis(aminoethylether)N,N, N',N'-tetraacetate egl egg laying-defective EJCs excitatory junctional currents Emc expulsion muscle contraction FHM familial hemiplegic migraine fir fluoride resistant g gram glr glutamate receptor family (AMPA) x g x gravity GABA Y-aminobutyric acid GFP green fluorescent protein HEK human embryonic kidney HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid hr(s) hour(s) HVA high voltage-activated HypoPP hypokalemic periodic paralysis HYPP hyperkalemic periodic paralysis xvii IP3 inositol 1,4,5-triphosphate itr inositol 1,4,5-triphosphate receptor K + potassium kb kilobase kDa kiloDaltons kg kilogram 1 liter . X dil X dilution buffer LVA low voltage-activated M molar max maximum mCi milliCurie MCS multiple cloning site mGluRl metabotropic glutamate receptor, type-I mg milligram min minute(s) ml milliliter mm millimeter mM millimolar MD megaohms Muv multiyulva Na + sodium NAChR nicotinic acetylcholine receptor nca novel four domain-type VGIC oti subunit ng nanogram xviii NGM nematode growth media N i 2 + nickel NLS nuclear localization signal NMJ neuromuscular junction co-Aga co-agatoxin co-CgTx co-conotoxin GVIA (o-CTx co-conotoxin PAGE polyacrylamide gel electrophoresis Pat paralyzed arrest at embryonic two-fold stage PBS phosphate-buffered saline pBoc posterior body muscle contraction PCR polymerase chain reaction PKA cAMP-dependent protein kinase PKC protein kinase C PMC paramyotonia congenita pmol picomole RACE rapid amplification of cDNA ends rad radiation absorbed dose ric resistant to inhibitor of cholinesterases RNA ribonucleic acid rpm revolutions per minute RT-PCR reverse transcription-polymerase chain reaction s second(s) SCA-6 spinocerebellar ataxia type-6 SDS sodium dodecyl sulfate xix SNARE soluble N-ethylmaleimide-sensitive attachment factor receptor SNAP-25 synaptosomal protein of 25 kDa snb synaptobrevin snt synaptotagmin str seven transmembrane receptor U units |ng microgram lull microliter urn micrometer uM micromole us microseconds unc uncoordinated UTR untranslated region V volts VGIC voltage-gated ion channel vol volume x times X X Acknowledgements I would like to express my sincerest thanks to my supervisor, Dr. Terrance Snutch, for his guidance, support and patience. I would also like to thank past and present members of the Snutch laboratory, including Dr. Colin Thacker, Dr. Esperanza Garcia, Dr. John McRory, Dr. Celia Santi, Dr. Donald Nelson, Dr. Eleanor Mathews, Dr. Heather Guthrie, Tracy Evans, Matthew Dwinnell and Michael Hildebrand. I would like to thank Dr. Michael Crowder and Laura Metz at the Washington University, St. Louis for their expertise and dedication to the halothane assay performed in this study and to Dr. David Miller and Joseph Watson at the Vanderbilt University for their help in identifying the motor neuron cell bodies present in the ventral nerve cord. I would also like to thank Dr. Catharine Rankin at the University of British Columbia for the use of her laboratory facilities and Ravinder Pandher for her help in scoring the thrashing assays. Finally, I would like to thank my friends, colleagues and mentors at the University of British Columbia, including Dr. Mary Gilbert, Dr. Jacob Hodgeson, past and present members of the Auld, Moerman and Roskams laboratories and my supervisory committee: Dr. Vanessa Auld, Dr. Donald Moerman and Dr. Lynn Raymond. xxi Dedication This thesis is dedicated to my wife, Cecilia M. Hamming and to my son, Sebastian A. Hamming, whose constant love and unconditional support helped me to gather the strength and courage needed to succeed. Also, a special thank you to all my extended family and friends who helped me out along the way. xxii Chapter 1. Introduction Electrical signaling within the nervous system depends upon rapid changes in the electrical potential across the cell membrane. These changes in potential occur in response to ions flowing quickly across the membrane through the pores of integral membrane proteins called ion channels. As a consequence of this electrical flow of ions, these channels are involved in numerous physiological processes including establishing and maintaining the membrane potential, regulating cellular excitability, generating and propagating action potentials and the release of neurotransmitters and hormones (Hille, 2001). Previous electrophysiological studies in neurons and muscles have revealed that the diversity of the physiological processes mediated by ion channels is encoded by a variety of different types of native ion channels that can be distinguished from one another based upon their ion selectivity, electrophysiological properties, mode of activation and sensitivity to different blocking agents (Hille, 2001). For organizational purposes, ion channels have been grouped into two major classes based upon the type of signal that controls their opening and closing. The first major class of ion channels is the ligand-gated ion channels that are primarily activated by different types of signaling molecules including neurotransmitters, nucleotides and intracellular calcium (Ca ). In contrast, the second major class of ion channels, called voltage-gated ion channels (VGICs), is primarily activated by changes in membrane potential. This grouping of ion channels into classes based upon their mode of activation is not absolute as there 1 are ligand-gated ion channels that are also sensitive to voltage and some VGICs may also require second messengers for activation (Hille, 2001). There is considerable functional and molecular diversity amongst VGICs and the remainder of the introduction will concentrate on just a subset of VGICs called the four domain-type VGICs. Evolution of Four Domain-Type Voltage-Gated Ion Channels While VGICs are complex proteins, they all contain a relatively simple motif that consists of at least two transmembrane segments separated from one another by a pore-forming loop (P-Loop) (Figure 1 A). This basic protein motif constitutes the entire channel in the mammalian family of inward rectifying potassium (K*) channels (Doupnik et al., 1995) and is thought to have served as the basic building block for the VGIC superfamily (Anderson and Greenberg, 2001). For example, through the acquisition of four additional transmembrane segments, the basic two transmembrane motif was transformed into an ion channel consisting of six transmembrane segments akin to what is found in the four domain-type voltage-gated K + , Ca2+and sodium (Na+) channels (Anderson and Greenberg, 2001) (Figure 1A). Molecular cloning and sequencing of the first Na + (Noda et al., 1984), Ca 2 + (Tanabe et al., 1987) and K + (Kamb et al., 1987; Papazian et al., 1987; Pongs et al., 1988; Schwarz et al., 1988) channels and the subsequent comparison of their amino acid sequences revealed common structural and organizational features amongst these ion channels and confirmed an evolutionary relationship between these functionally diverse channels (Catterall, 1988; Jan and Jan, 1990). These comparisons showed that the pore-forming units of K + , Ca 2 + and Na + channels all contain a basic protein motif consisting of six transmembrane segments, called S1-S6, and that the S4 segment contains regularly repeated positive residues and is believed to form part of the voltage-sensing mechanism of the channel. Furthermore, the two hydrophobic segments between the S5 2 and S6 transmembrane segments, called SSI and SS2 (or P-Loop), are proposed to line the pore of the channel and are thought to be responsible for its ion selectivity properties (Figure 1 A). In order to form functional channels four of these basic units, called domains, must associate together. For K + channels, each of these domains is a separate protein, whereas for Ca 2 4 and Na + channels a single large protein encompasses all four domains. The difference in the subunit structure between K + channels and that of Ca 2 + and Na + channels suggested that a common ancestral single-domain channel, similar to present day K + channels, gave rise to the ancestor of the Ca 2 + channel by two rounds of gene duplication followed each time by sequence divergence and that Na + channels arose from Ca 2 + channels by additional divergence from the ancestral Ca 2 + channel gene (Hille, 1989) (Figure IB). This proposed pattern of gene duplication is supported by the observation that in both Ca 2 + and Na 2 + channels, domains I and HI are more similar to one another than either are to domains II and IV and that these latter two domains are themselves more similar to one another (Strong et al., 1993). Furthermore, this proposed model of gene duplication implies the existence of a two-domain protein that was capable of forming a functional channel and served as the intermediate channel between the single and four-domain channels (Figure IB). The expected existence of a two-domain channel has been confirmed by the cloning of the TPC1 (two-pore channel i) protein from rat kidney (Ishibashi et al., 2000) and by the identification of another two-domain protein from Arabidopsis (AAD11598) reported only in GenBank (Anderson and Greenberg, 2001). The hypothesis that Na + channels arose from Ca 2 + channels as a result of subsequent sequence divergence from their common ancestral four-domain channel is based upon surveying the distribution of native Ca 2 + and Na + channels in lower eukaryotes organisms. In general, protozoans use Ca 2 + and not Na + as the primary inward charge carrier and it is not until early metazoans, such as the cnidarians, that purely Na+-dependent action potentials are observed 3 Figure 1. Diagrams Representing the Proposed Evolutionary Events that Have Given Rise to the Four Domain-Type Families of VGICs A) A depiction of the structural motifs that gave rise to the diversity of VGICs. All VGICs contain a relatively simple motif (1 domain 2-TM) that consists of at least two transmembrane segments separated from one another by a pore-forming loop (blue). Through the acquisition of four additional transmembrane segments to the amino terminus of the 1 domain 2-TM protein, the basic two transmembrane motif was transformed into an ion channel consisting of six transmembrane segments (1 domain 6-TM) similar to modern day K + channels. The S4 voltage sensor is indicated in red. (Figure adapted from Anderson and Greenberg, 2001). B) A schematic diagram representing the two rounds of gene duplication thought to have given rise to modern day Ca 2 + and Na+ channels. According to this model, a common ancestral single-domain channel gene (A) was duplicated to create a two-domain structure (A | A) that subsequently underwent sequence divergence. This divergent two-domain structure (A | B) was once again duplicated and gave rise to a precursor four-domain structure (A | B | A | B). This precursor structure underwent further sequence divergence and gave rise to the four domain-type ion channel structures seen today (I | II | III | IV). According to this model, the sequences of domains I and III and domains II and IV of Ca 2 + and Na + channels should be more similar to one another due to their close evolutionary history (Figure taken from Strong et al., 1993). 4 A 1 domain 2-TM +HJV out CO, 1 domain, 6-TM out in B DUPUCATION DIVERGENCE DUPLICATION B A B A B DIVERGENCE I II III IV (Hille, 1984; Anderson, 1987; Anderson and Greenberg, 2001). Based upon the observed distribution of Ca 2 + and Na + currents in lower eukaryotes, Hille (1984) hypothesized that Na+ channels evolved from Ca 2 + channels along with the evolution of the first true nervous systems. The requirement for Na + as the major charge carrier may have been a necessary development because if Ca 2 + was used as the sole internal charge carrier, intracellular Ca 2 + concentrations could reach toxic levels under conditions of high neuronal activity. This hypothesis was formulated before the cloning of Ca 2 + or Na + channels and is supported by computer-based evolutionary analysis programs using the sequences of the currently identified Ca 2 + and Na + channel families described in the following sections (Anderson and Greenberg, 2001). Four Domain-Type Voltage-Gated Ion Channels Calcium Channels The major pathway for the rapid entry of Ca 2 + into excitable cells is mediated by a heterogeneous family of proteins called voltage-gated Ca 2 + channels. Ca 2 + entry into cells mediates a number of physiological processes including neurotransmitter release, excitation-contraction coupling, regulation of gene expression, activation of Ca2+-dependent enzymes, modulation of ion channel activity and cell migration (Tanabe et al., 1988; Catterall, 1991; Komuro and Rakic, 1992; Uchitel et al., 1992; Bading et al., 1993; Wheeler et al., 1994). Furthermore, the amount and timing of Ca 2 + influx can be directly modulated by protein kinases and phosphatases, as well as by a variety of neurotransmitters, neuropeptides and hormones acting on the Ca 2 + channels through a variety of second messenger pathways (Catterall, 2000b). Electrophysiological recordings from neurons, muscles and endocrine cells have revealed that there are a number of different Ca 2 + conductances indicating the existence of multiple types of Ca 2 + channels (Nowycky et al., 1985; Tsien et al., 1988; Bean, 1989; Hess, 1990; Llinas et al., 6 1992). Based upon these early studies, Ca 2 + channels were categorized as either low-voltage activated (LVA or T-type) or high-voltage activated (HVA) based upon the membrane potential at which they first open. The LVA Ca 2 + channels typically have small conductances, are activated by small depolarizations from hyperpolarized membrane potentials, rapidly inactivate in a voltage-dependent manner and are sensitive to Ni 2 + , octanol and amiloride (Bean, 1989; Tsien et al., 1991). In contrast, HVA Ca 2 + channels are activated by stronger depolarizations and display variable inactivation kinetics (Jones, 1998). Using other electrophysiological and pharmacological criteria, vertebrate HVA Ca channels have subsequently been further subdivided into several different types: L-, N-, P-, Q- and R-type (De Waard et al., 1996). L-, N-, P-, Q- and R-type Ca 2 + channels in vertebrates are largely distinguished from one another on the basis of their pharmacological properties. L-type Ca 2 + channels are sensitive to 1,4-dihydropyridines (DHPs) and phenylalkylamines (Reuter, 1983; Bean, 1989). N-type Ca 2 + channels are resistant to DHPs, but are irreversibly blocked by ©-CgTx GVTA, a peptide toxin from the marine snail, Conus geographus (Olivera et al., 1984; McCleskey et al., 1987; Tsien et al., 1988). P-type Ca 2 + channels are not affected by either DHPs or ©-CgTx GVIA, but are blocked at low concentrations of ©-Aga IVA, a peptide toxin from the funnel web spider Agelenopsis aperta (Mintz et al., 1992). Q-type Ca 2 + channels are unaffected by either DHPs, ©-CgTx GVIA or ©-Aga IVA at concentrations that block L-, N- and P-type Ca 2 + channels, respectively. However, Q-type Ca 2 + channels are blocked by co-Aga IVA at concentrations 10 to 20 times greater than that required to block P-type Ca 2 + channels and are completely blocked by co-CTx MVIIC, a peptide toxin derived from the marine snail Conus magus (Olivera et al., 1994). R-type Ca 2 + channels represent all those Ca 2 + conductances that are HVA and are not blocked by DHPs, ©-CgTx GVIA, ©-Aga IVA or ©-CTx MVIIC (Zhang et al., 1993), but are 7 specifically blocked by the synthetic peptide toxin SNX-482 derived from the venom of the tarantula, Hysterocrates gigas (Newcomb et al., 1998). Molecular genetic and biochemical studies of Ca 2 + channels have shown that HVA Ca 2 + channels are mulitprotein complexes consisting of three or four subunits; the ai, 0:28, (3 and/or y subunits (Takahashi et al., 1987; Catterall, 2000b; Kang et al., 2001; Arikkath and Campbell, 2003) (Figure 2 A). The pore-forming cti subunit (190 to 250 kDa) is the largest subunit of the Ca 2 + channel complex and consist of four homologous, mainly hydrophobic domains (designated domains I-IV) each comprised of six putative membrane-spanning segments (S1-S6). The S4 segment in each domain contains positively charged residues in every third or fourth position (Tanabe et al., 1987; Ellis et al., 1988) and together are believed to form part of the voltage-sensing mechanism of the channel (Armstrong, 1981; Catterall, 1986; Kontis et al., 1997). Between the S5 and S6 transmembrane segments of each domain are two hydrophobic segments, SSI and SS2 (or P-Loop), which are predicted to associate together to form the pore of the channel (Guy and Conti, 1990) (Figure 2B). To date, ten different Ca 2 + channel ai subunit genes have been identified in mammals (otiA-i and ais) and multiple alternatively spliced isoforms of each ai subtype exist (Hofmann et al., 1999). Whole cell and single channel electrophysiological recordings in Xenopus oocytes and mammalian cell lines have provided information about the functional and pharmacological properties of cloned Ca 2 + channel ai subunits. The results show that the a i A subunit encodes for both P- and Q-type Ca 2 + channels (Cav2.1), the CXIB subunit encodes an N-type Ca 2 + channel (Cav2.2), the ais, die, am, and aiF subunits encode L-type Ca 2 + channels (Cavl.l-Cav1.4, respectively), the am, am, and an subunits encode T-type Ca 2 + channels (Cav3.1-Cav3.3, respectively), and the aiE subunit encodes a novel class of HVA Ca 2 + channel (Cav2.3) with some properties similar to R-type Ca 2 + currents (Hofmann et al., 1999). Alignment of the amino 8 Figure 2. Composition of a H V A Ca Channel Complex and Structure of the a i Subunit A) A diagram of the H V A C a 2 + channel complex which consists of four separate protein subunits; the a i , 0:28, P and y subunits. The a i subunit is thought to form the channel proper of the complex and contains both the Ca2+-selective pore and the voltage-sensing mechanism. The intracellular {3 subunit and the membrane attached 0:28 and y subunits interact with the a i subunit and modulate its electrophysiological properties and/or facilitate its expression. B) The predicted structure and transmembrane topology of the C a 2 + channel a i subunit. The a i subunit is predicted to consist of four homologous, mainly hydrophobic domains (designated domains I-TV) each comprised of six putative membrane-spanning segments (S1-S6) (cylinders). The S4 segments in each domain contain positively charged residues (indicated by +) and together are believed to function as the voltage sensor of the channel. Between the S5 and S6 transmembrane segments of each domain are two hydrophobic segments, SSI and SS2 (or P-Loop), which are predicted to associate together to form the pore of the channel. 9 A Ca 2* • • B I II III IV +NH3 COO-10 acid sequences encoding the transmembrane domains of the different mammalian Ca channel ai subunits reveals that there are three distinct subfamilies (Cavl to Cav3) of Ca 2 + channels based upon sequence identities. Members within a subfamily are greater than 70% identical with one another, whereas members of one subfamily are less than 50% identical to members of another subfamily (Ertel et al., 2000). This pattern of Ca 2 + channel subfamily grouping agrees with previous Ca channel groupings based upon electrophysiological and pharmacological criterion (Figure 3). The p subunit has been the most extensively studied of the Ca 2 + channel accessory subunits and appears to have the most profound effect on the physiological properties of the c*i subunit. Analyses of the amino acid sequence of the different P subunits have suggested that there are no transmembrane segments or glycosylation sites within these proteins, consistent with the hypothesis that the P subunit is a cytoplasmic subunit of the HVA Ca 2 + channel complex (Ruth et al, 1989; Pragnell et al., 1991; Hullin et al., 1992; Perez-Reyes et al., 1992; Castellano et al., 1993). In mammals, there are at least four different P subunits (pi to P4) encoded by distinct genes, with alternative splicing generating additional diversity (Ruth et al., 1989; Hullin et al., 1992; Perez-Reyes et al., 1992; Castellano et al., 1993; Helton and Home, 2002; Arikkath and Campbell, 2003). Although the specific effects of channel modulation depend upon the p subunit isoform, all p subunits appear to have the same general impact on the properties of HVA Ca channel cti subunits. Coexpression of a P subunit with various cti subunits increases whole-cell currents, most likely by promoting the insertion of the cti subunit into the membrane and/or by facilitating channel opening (Singer et al., 1991; Neely et al., 1993; Hofmann et al., 1999). In addition, coexpression of the P subunit alters channel kinetics. With the exception of the P2 subunit, which slows inactivation and causes a positive shift in the steady-state inactivation 11 Figure 3. Identity Tree Of Mammalian Ca Channel a i Subunits The predicted amino acid sequences of mammalian representatives of each Ca 2 + channel ai subunit were compared pairwise using the CLUSTAL W algorithm and the percent identities were plotted. Only the amino acid sequences of the four conserved domains were used. GenBank Accession Numbers: rat aXA, M64373; rat am, M92905; rat otic, M67515; rat am AF370009; rat a ] E , L15453; human aiV, AJ224874; rat a 1 G , AF290212; rat a m , AF290213; rat a n , AF290214 and rabbit ais, M23919. 12 L-Type H V A Non L-Type Cavil (ais) Cavl.2 (aic) Cavl.3 (a ]D) Cavl.4 (aiF) Cav2.1 (aw) Cav2.2 (am) Cav2.3 (am) L V A T-Type Cav3.1 (aio) Cav3.2 (am) Cav3.3 (a n) I 1 1 [ 20 40 60 80 100 Percent Identity 13 curve, all other p subunits increase the rate of channel activation and inactivation and shift the steady-state inactivation curve to hyperpolarized potentials (Singer et al., 1991; Hullin et al., 1992; Castellano et al., 1993; Hofmann et al., 1999). The p subunit is also involved in the trafficking of the a, subunit to the membrane, as binding of the P subunit to the domain I-II linker antagonizes the endoplasmic reticulum retention signal present in that region of the ai subunit (Bichet et al., 2000). The Ca channel 0:2 and 5 subunits are transcribed from the same gene and are proteolytically cleaved in vivo to generate the 143 kDa 0:2 subunit and the 27 kDa 8 subunit which are subsequently linked by disulfide bonds (De Jongh et al., 1990; Kim et al., 1992). Both the ct2 and 8 subunits are heavily glycosylated suggesting that the two subunits are mainly extracellular except for a single transmembrane segment present in the 8 subunit that presumably anchors the 0128 complex to the membrane (Gurnett and Campbell, 1996). In mammals, four different a 28 genes have been identified (0:28-1 to a28-4) (Klugbauer et al., 1999; Gao et al., 2000; Qin et al., 2002) with alternative splicing generating further diversity (Arikkath and Campbell, 2003). The functional effects of the 0C2S are subtler than that of the P subunit and depend upon the class of oti subunit and the cell type used for expression. The 0:28 may be required for efficient expression and/or trafficking of the ai subunit to the membrane (Mikami et al., 1989). Amino acid sequence analyses of the different Ca 2 + channel y subunits predict that they are glycoproteins consisting of four membrane-spanning domains. The y subunit family is encoded by at least eight different genes (yi to y») (Bosse et al, 1990; Eberst et al., 1997; Letts et al., 1998; Klugbauer et al., 2000; Arikkath and Campbell, 2003). Coexpression of different y subunit with the a i , 0:28 and p subunits alters the kinetics of activation and inactivation to varying degrees (Arikkath and Campbell, 2003). 14 Sodium Channels The major roles of voltage-gated Na + channels are to mediate rapid membrane depolarization, propagate the action potential down the length of the axon and trigger neurotransmitter release or hormone secretion through the activation of voltage-gated Ca 2 + channels. The amount of Na + influx through Na + channels and their corresponding ability to generate action potentials can be directly modulated by a variety of different protein kinases (Costa and Catterall, 1984a, b; Li et al., 1992; Smith and Goldin, 1996; Hilborn et al., 1998). Although recordings of native Na + currents from different tissues and species reveals that Na + channels exhibit a range of electrophysiological properties, they do not show the diversity that is characteristic of the Ca 2 + channel family (Goldin, 2001). It appears that it is the small differences in the properties among different Na + channels that contribute to their specialized physiological roles. Na + channels from different tissues and species have historically been distinguished from one another based upon their sensitivities to various toxins (Catterall, 1980; Anderson, 1987), as well as by subtle differences in their electrophysiological properties (Mandel, 1992). For these reasons, Na + channels have traditionally been classified based upon the source of the channel. Molecular genetic and biochemical studies of Na + channels have shown that they are mulitprotein complexes consisting of a highly processed a subunit (-260 kDa) and either one or two accessory (3 subunits (33 to 36 kDa) depending upon the tissue in which the Na + channel is expressed (Catterall, 2000a) (Figure 4). The a subunits of Na + channels, like the oil subunits of Ca 2 + channels, are the pore-forming subunit of the Na + channel complex and contain all of the essential elements necessary for Na + channel function, including channel activation (opening), ion selectivity and rapid inactivation (closing). Similar to Ca 2 + channels, the Na + channel a subunits consist of four homologous, mainly hydrophobic domains (designated domains TIV), each comprised of six putative membrane-spanning segments (S1-S6) including S4 segments containing positively charged residues in every third or fourth position (Armstrong, 1981; Noda 15 et al., 1984; Catterall, 1986; Kontis et al., 1997). The domain IJJ-IV linker serves as the inactivation gate, blocking the channel pore during sustained depolarizations of the membrane (Vassilev et al., 1988). Between the S5 and S6 transmembrane segments of each domain are two hydrophobic segments, SSI and SS2 (or P-Loop), which are predicted to associate together to form the pore of the channel (Guy and Conti, 1990) (Figure 4). To date, nine different Na + channel a subunit genes (Navl.l to Nav1.9) have been identified in mammals and alternatively spliced isoforms of some of the a subunits exist (Goldin, 2001). Alignment of the amino acid sequences encoding the transmembrane domains of the nine different rat Na + channel a subunits reveals that they are greater than 50% identical to one another (Figure 5). Unlike the Ca 2 + channel family which has three subfamilies, the nine Na + channels are considered to members of a single family (Catterall et al., 2003). In addition to the nine Na + channels that have been functionally expressed in exogenous systems, another putative a subunit gene called, Nax, has been identified and is a more divergent class of Na + channel because it is only approximately 50% identical to the other Na + channel a subunits (George et al., 1992; Catterall et al., 2003). The Nax channel subtype has yet to be functionally expressed (George et al., 1992). Na + channel a subunits in mammals are associated with either one or two accessory p subunits depending upon where the Na + channel is expressed. In mammals, there are at least three different P subunits (Pi, p 2 and p3) encoded by distinct genes, with alternative splicing occurring for Pi (Isom et al., 1994; Isom et al., 1995; Isom and Catterall, 1996; Morgan et al., 2000; Goldin, 2001). Na + channels in the adult central nervous system (CNS) contain Pi (or p3) and p2 subunits, whereas Na + channels in the adult skeletal muscle contain only a p2 subunit. Biochemical analysis of the Na + channel complex has revealed that Pi subunits are noncovalently associated with the a subunit, whereas p 2 subunits are covalently linked to the a subunit by a 16 Figure 4. Composition of a Na+ Channel Complex and Structure of the a Subunit The primary structure of the Na + channel a subunit is predicted to consist of four homologous, mainly hydrophobic domains (designated domains I-IV) each comprised of six putative membrane-spanning segments (S1-S6) (cylinders). The S4 segments in each domain contain positively charged residues (indicated by +) and together are believed to function as the voltage sensor of the channel. Between the S5 and S6 transmembrane segments of each domain are two hydrophobic segments, SSI and SS2 (or P-Loop), which are predicted to associate together to form the pore of the channel. The sites of PKA (P in a circle) and PKC (P in a diamond) phosphorylation and the inactivation particle (h in a circle) within the domain LTJ-IV linker are shown. The extracellular domains of the Pi and p 2 subunits are illustrated as immunoglobulin-like folds and the predicted interaction site between the a and Pi subunits is shown. (Figure adapted from Catterall, 2000a). 17 18 Figure 5 . Identity Tree Of Rat Na+ Channel a Subunits The predicted amino acid sequences of each rat Na + channel a subunit were compared pairwise using the CLUSTAL W algorithm and the percent identities were plotted. Only the amino acid sequences of the four conserved domains were used. GenBank Accession Numbers: rat Na vl. 1, X03638; Nav1.2, X03639; Nav1.3, Y00766; rat Nav1.4, M26643; rat Nav1.5, M27902; rat Nav1.6, AF049239; rat Nav1.7, AF000368; rat Nav1.8, X92184 and rat Nav1.9, AJ237852. 19 I Navl.l I Navl.2 I Navl.3 Nav1.6 I Navl.7 I Navl.4 I Nayl.5 I Nayl.8 Navl.9 —I 1 1 60 80 100 Percent Identity 20 disulfide bond (Messner and Catterall, 1985). Analysis of the amino acid sequences of the different P subunits predicts a protein with a single membrane-spanning region and that also has immunoglobulin-like folds similar to those found in neural cell adhesion molecules (Isom et al., 1995; Isom and Catterall, 1996; Morgan et al., 2000). The effect of coexpression of a P subunit with an a subunit on Na + channel electrophysiological properties depends upon the P subunit. In Xenopus oocytes, coexpression of the Pi subunit leads to an increase in the rate of inactivation and causes a negative shift in the steady-state inactivation profile (Isom et al., 1992; Smith and Goldin, 1998), whereas coexpression of the p3 (Morgan et al., 2000) or p2 (Isom et al., 1995) subunits modulate Na + channel gating to a lesser extent. In addition to modulating the gating kinetics of the a subunit, the p2 subunit may also be involved in inserting channels into the membrane (Schmidt et al., 1985). Furthermore, presumably through their immunoglobulin-like folds, the Pi and P2 subunits have been shown to interact with extracellular matrix proteins and thus may function as cellular adhesion molecules (Srinivasan et al., 1998). Ion Channelopathies of Four Domain-Type Voltage-Gated Ion Channels The examination of naturally occurring disease-causing mutations in four domain-type VGICs has fostered new insights into the biological roles and physiological functions of these ion channels by providing a better understanding of the relationships between structure and function. Likewise, analysis of the behavioral defects exhibited by humans and animals exhibiting mutations in Ca 2 + and Na + channels can further the present knowledge of cellular and systems biology by helping to draw connections between the mutant behavior exhibited (phenotype) and the cellular localization of the affected protein. 21 Calcium Channelopathies In addition to the normal physiological functions mediated by Ca 2 + channels, they are also implicated in a number of human diseases including night blindness, migraine headaches and certain types of movement and muscle disorders (Snutch and Reiner, 1992; Lorenzon and Beam, 2000; Pietrobon, 2002). The physiological disturbances presumed to be the result of mutations to Ca 2 + channels are quite different from those caused by mutations to Na + channels. The study of the physiological basis of a number of Na + channelopathies has revealed that these disease states can be attributed to altered electrical excitability of the affected cell which can be easily understood in terms of changes to the electrophysiological properties of the mutant Na + channels (see below; Sodium Channelopathies). In contrast, because Ca 2 + plays an important role in intracellular signaling, mutations to Ca 2 + channels may result in widespread changes to biological processes within the affected cell that may be the result of only minor changes to electrophysiological properties. For example, because the concentration of intracellular Ca 2 + is so tightly regulated, mutations to Ca 2 + channels may cause cytotoxicity to neurons either by increasing (Choi, 1988) or decreasing (Koike etal., 1989; McCaslin and Smith, 1990; Koh and Cotman, 1992) intracellular Ca 2 + levels and this cellular atrophy may be the pathophysiological cause of the disease condition (Lorenzon and Beam, 2000). ais Subunit Previous research on Ca 2 + channelopathies in both humans and mice have implicated a number of different Ca 2 + channel ai and accessory subunits in disease states (Snutch and Reiner, 1992; Lorenzon and Beam, 2000; Pietrobon, 2002). For example, mutations in the Ca 2 + channel gene involved in excitation-contraction coupling in human muscle have been shown to cause hypokalemic periodic paralysis (HypoPP), a dominant disorder characterized by episodic muscle weakness. Molecular genetic analysis of patients with HypoPP has revealed that this disorder is 22 associated with three different missense mutations in the voltage-sensing S4 segments of the otis subunit (Jurkat-Rott et al., 1994; Ptacek et al., 1994; Fouad et al., 1997). Similarly, the muscular dysgenesis mouse mutant contains a truncation mutation in the ocis subunit of the skeletal muscle L-type Ca 2 + channel that causes a lethal phenotype (Beam et al, 1986; Tanabe et al., 1988; Knudsonetai., 1989). aw Subunit Missense, nonsense and deletion mutations to the human a i F subunit have been implicated in incomplete X-linked congenital stationary night blindness (Bech-Hansen et al., 1998; Strom et al., 1998). This condition is characterized by impaired vision at night, as well as by variable daytime vision and is presumed to be the result of altered neurotransmitter release from the photoreceptor cells to the second-order neurons due to alterations in the electrophysiological properties of the C I I F L-type Ca 2 + channel (Schmitz and Witkovsky, 1997; Bech-Hansen et al., 1998; Strom et al., 1998; McRory et al, 2004). With the recent molecular cloning of the human CXIF subunit, the effects of these mutations on channel function are beginning to be addressed (McRory et al., 2004). a1A Subunit Mutations to the human c t iA subunit cause several autosomal-dominant neurological disorders in humans including familial hemiplegic migraine (FHM), episodic ataxia type-2 (EA-2) and spinocerebellar ataxia type-6 (SCA-6), all of which have abnormal cerebellar function and/or atrophy as a part of their pathophysiology (Ophoff et al., 1996; Zhuchenko et al., 1997; Pietrobon, 2002). Since the CCIA subunit is highly expressed in the neurons of the cerebellum (Westenbroek et al., 1995),.it is not surprising that mutations to the CXIA subunit cause significant 23 movement disorders. As the name of the disorder suggests, FHM is a rare subtype of migraine with aura that is accompanied by weakness or paralysis of one side of the body that typically lasts for hours up to days (Lorenzon and Beam, 2000; Pietrobon, 2002). In addition, there may be accompanying cerebellar atrophy and the onset of FHM is generally during childhood or early adolescence. Molecular genetic studies of families with FHM have revealed that the disorder is the result of missense mutations that alter highly conserved amino acid residues in various parts of the channel important for function (Ophoff et al., 1996; Lorenzon and Beam, 2000; Pietrobon, 2002). A number of the mutations have been introduced into cloned CXIA subunits and the electrophysiological properties of the mutant channels tested in surrogate expression systems. The conclusions drawn from these studies are that by changing the voltage-dependent or kinetic properties of the channels to varying degrees, some mutations decrease the amount of Ca 2 + entry whereas others cause an increase in Ca 2 + entry (Kraus et al., 1998; Hans et al., 1999). EA-2 is a rare autosomal-dominant disorder characterized by ataxia, nystagmus, dysarthria and vertigo which may be triggered by fatigue or stress (Lorenzon and Beam, 2000; Pietrobon, 2002). The onset of EA-2 is in late childhood or during adolescence and cerebellar atrophy usually is prevalent in this disorder. In addition to the aforementioned conditions, approximately half of EA-2 patients also report having migraine-like symptoms. Molecular genetic analyses have revealed that the majority of the mutations identified from patients with EA-2 disrupt the reading frame of the CXIA subunit leading to a truncated protein product. In contrast, relatively few mutations have been found that are the result of missense mutations (Pietrobon, 2002). One of the identified missense mutations has been characterized in a recombinant human aiA channel and leads to the complete loss of channel function without altering its level of expression (Guida et al., 2001). Based upon the observation that the aforementioned mutations probably result in the production of either truncated or full-length non-functional channels, the prevailing explanation for the autosomal dominance of the EA-2 24 disorder is either one of haploinsufficiency or that the truncated channels serve as dominant-negative proteins that compete for channel accessory or regulatory proteins (Lorenzon and Beam, 2000). The autosomal-dominant disorder SCA-6 is characterized by ataxia, nystagmus, dysarthria, and neuronal loss in the cerebellum (Lorenzon and Beam, 2000; Pietrobon, 2002). In contrast to FHM and EA-2, the age of SCA-6 onset is usually between the ages of 40 to 50. Molecular genetic analyses of patients with SCA-6 reveal that this disorder is due to a CAG trinucleotide repeat expansion that introduces polyglutamines approximately 100 amino acids from the carboxy terminus of the OCIA subunit (Zhuchenko et al., 1997). Furthermore, it has been found that increasing the size of the CAG expansion has been correlated with an earlier onset and increased severity of the disease. Similar to other trinucleotide repeat diseases, the pathophysiology of SCA-6 may be the result of altered stability of the mutant channel protein or may be the result of an alteration of the biophysical properties of the CXIA subunit (Matsuyama et al., 1999; Restituito et al., 2000; Torn et al., 2000). In addition to the aforementioned diseases attributed to mutations to the human CXIA subunit gene, four different mouse strains have also been found to show behavioral phenotypes that have been linked to mutations in the CXIA subunit gene. Tottering mice display seizures and mild ataxia as a result of a missense mutation in the linker between transmembrane segments S5 and S6 of domain II (Fletcher et al., 1996; Doyle et al., 1997). The tottering mutation is believed to decrease the number of functional channels that are expressed in the cell membrane as Ca 2 + channel current densities are significantly reduced in tottering mice, as well as in tissue culture cells expressing the mutant channel (Wakamori et al., 1998). Leaner mice also have a defect in the a iA subunit gene and show severe ataxia and cerebellar atrophy similar to the human ataxic disorders, EA-2 and SCA-6 (Fletcher et al., 1996; Doyle et al., 1997). Both result in a decrease in 25 Ca z + channel current density in Purkinje cells (Dove et al., 1998). The nature of the mutation in leaner mice has been determined to be a single nucleotide substitution in a splice donor consensus sequence of the ai A subunit gene that results in a change to the reading frame and produces two aberrant isoforms with altered carboxy terminal sequences of different lengths (Wakamori et al., 1998). The remaining two mouse strains, rocker (Zwingman et al., 2001) and roller (Mori et al., 2000), are also caused by missense mutations in CXIA subunit gene. Accessory Subunits Mouse strains with mutations to the HVA Ca 2 + channel accessory subunits have also been identified. An insertion of four basepairs in a splice donor site of the P 4 subunit gene has been found to eliminate the expression of this accessory protein and cause the lethargic disorder in mice (Burgess et al., 1997). Lethargic mice display ataxic movement disorders and have seizures that mimic that seen in the human GCIA subunit gene disorders described above but lack the associated neuronal degeneration (Dung and Swigart, 1972; Burgess et al., 1997). In stargazer mice, expression of the 72 subunit gene is disrupted by a transposon insertion in the first intron and misexpression of the 72 accessory subunit is responsible for ataxic movement and epileptic seizures (Noebels et al., 1990; Letts et al., 1998). Although, recent studies involving stargazin have shown that this protein is involved in the functional expression of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and acts as an accessory subunit for these channels (Tomita et al., 2003; Tomita et al., 2004). Sodium Channelopathies In addition to the normal physiological functions mediated by Na + channels, they are also implicated in a number of disorders of hyperexcitability in humans. For example, mutations in 26 the skeletal muscle Na + channel a subunit (Nav1.4) have been shown to cause different types of neuromuscular diseases (Cannon, 1997). The autosomal dominant neuromuscular diseases hyperkalemic periodic paralysis (HYPP) (Ptacek et al., 1991; Rojas et al, 1991) and paramyotonia congenita (PMC) (McClatchey et al., 1992; Ptacek et al., 1992) are the result of missense mutations that alter either the activation or inactivation properties of the skeletal muscle Na + channel leading to periodic paralysis (Cannon, 1997). Both HYPP and PMC are dominant mutations since only a small number of non-inactivating Na + channels are required to cause the sustained muscle depolarization typical of these disorders. Similarly, mutations in the cardiac muscle Na + channel a subunit (Nav1.5) leads to long QT syndrome, another human disease of hyperexcitability (Kass and Davies, 1996). In long QT syndrome, the mutant cardiac Na + channels have altered inactivation properties that lead to a small non-inactivating Na+ current during the plateau phase of the cardiac action potential (Wang et al., 1995; Wang et al., 1996). The prolonged interval between the QRS and T waves in electrocardiograms of patients with long QT syndrome increases their risk for cardiac arrhythmias (Wang et al., 1995). Caenorhabditis elegans as a Genetic Model for the Study of Voltage-Gated Ion Channels In addition to studies examining exogenously expressed and native mammalian Ca 2 + and Na+ channels, there is a need to define the physiological roles of these channels using genetic model systems. The correlation of mutant phenotypes with defined alterations that affect electrophysiological and pharmacological properties may identify important structural components that might not be identified from studying naturally occurring mutations or those induced by site-directed mutagenesis based upon educated guesses. Furthermore, the genetic and molecular analysis of extragenic suppressors of Ca 2 + and Na+ channel mutants will both define important interactions between these ion channel subunit proteins and identify novel gene products that interact with these ion channel complexes. 27 The nematode Caenorhabditis elegans provides an ideal model system for studying ion channel functions in animals. C. elegans is useful for the genetic analysis of gene structure and function because mutations can be easily introduced and the hermaphroditic mode of reproduction facilitates the isolation and maintenance of mutant strains (Herman, 1988). Males only make up ~0.1% of wild-type populations (Hodgkin et al., 1979) and are used as a means of generating genetic diversity within natural populations and in the laboratory as a means of transferring genetic material between hermaphroditic strains. An extensive genetic map has been established for C. elegans and the entire genome has been cloned into YAC and cosmids vectors (Coulson et al., 1986; Coulson et al., 1988) which facilitates the genetic mapping of new mutations. In addition, genes from C . elegans can be manipulated by molecular techniques and can be reintroduced into the living organism by DNA transformation (Fire, 1986; Mello et al., 1991; Mello and Fire, 1995). The complete DNA sequence of the C . elegans genome has been determined ( C . elegans Sequencing Consortium, 1998) providing an unprecedented opportunity to explore not only the molecular neurobiology of this model organism and the physiology of other tissue systems, but the biology of the worm as a whole. Examination of the completed genome sequence of C. elegans has lead to the discovery of novel genes that were previously unidentifiable using classical genetic or conventional homology-based cloning techniques and has revealed that there is more diversity within well-characterized gene families, such as VGICs, than was previously thought (Bargmann, 1998). Furthermore, the study of these newly identified novel genes can be aided by the fact that the loss-of-function phenotype of any gene of interest in C. elegans can be determined using RNA interference (RNAi) (Montgomery et al., 1998; Timmons and Fire, 1998) and gene knockout methods (Zwaal et al., 1993; Jansen et al., 1997; Liu et al., 1999; Gengyo-Ando and Mitani, 2000). Efforts are presently underway to obtain null mutations in each of the 20,000 predicted genes in the C . elegans genome ( C . elegans Gene Knockout Consortium), 28 which will help to speed the elucidation of the biological functions and physiological roles of these new genes. C. elegans is a relatively simple organism consisting of only 959 somatic cells in the adult hermaphrodite. The lineage of each cell has been precisely determined and the number and position of each of these cells is essentially invariant (Sulston, 1983; Sulston et al., 1983). In spite of the relatively few numbers of cells, C. elegans possesses most of the tissue types seen in higher organisms, including epidermis, muscle and nervous tissues (White, 1988). The nervous system of C. elegans consists of 302 neurons and 56 support cells in the adult hermaphrodite and the connectivity and morphology of each of these neurons has been determined from serial section electron micrographs (White et al., 1986; Hall and Russell, 1991). The nervous system in C. elegans can be divided into two separate systems that are capable of functioning independently of one another. The first of these is the somatic nervous system which consists of 282 neurons and is responsible for generating the majority of behaviors (White et al., 1986). The second nervous system is the pharyngeal nervous system which contains the remaining 20 neurons and is responsible for generating and regulating feeding behavior (Albertson and Thomson, 1976; Avery and Thomas, 1997). Despite the relatively small size of the nervous system in C. elegans, it generates and regulates a repertoire of simple behaviors through the use of innate motor programs. Some of these behaviors include, locomotion, mechanosensation and touch avoidance, chemo and thermotaxis, egg-laying; mating, pharyngeal pumping and defecation. Behavioral paradigms have been developed to assess behavioral characteristics in both wild-type and mutant animals (Trent et al., 1983; Chalfie and White, 1988; Thomas, 1990) and have proven to be powerful experimental systems for the dissection of neural signaling pathways that underlie the execution of these behaviors. In addition, genetic, molecular and cell ablation techniques have helped to determine the physiological and behavioral roles of several classes of sensory and motor neurons 29 (Chalfie et al., 1985; Bargmann et al., 1993; Wicks and Rankin, 1995; Hobert et al., 1997). Since four domain-type VGICs are thought to be involved in a number of cellular processes including neurotransmission, muscle contraction, electrical signaling and cell migration, the availability of these simple behavioral assays should facilitate the analysis of behavioral defects in VGIC mutants. Voltage-Gated Ion Channel Genes in C. elegans Calcium Channels unc-2 Past molecular genetic techniques have been successful at identifying and behaviorally characterizing three different Ca 2 + channels genes in C. elegans. The first of these Ca 2 + channel genes identified by these approaches was the unc-2 gene which is predicted to encode the a.] subunit of a non L-type HVA Ca 2 + channel (Schafer and Kenyon, 1995). Behavioral analysis of unc-2 mutant animals reveals that unc-2 affects a wide variety of behaviors including movement (Brenner, 1974), pharyngeal pumping (Avery, 1993), egg-laying (Schafer and Kenyon, 1995) and defecation (Miller et al., 1996). A closer examination of the locomotion defect exhibited by unc-2 animals has revealed that unc-2 mutants are resistant to the acetylcholine esterase inhibitor, aldicarb, presumably due to a decrease in the amount of acetylcholine released at the neuromuscular junction, implicating UNC-2 in cholinergic transmission (Miller et al., 1996; Richmond et al., 2001; Mathews et al., 2003). In addition, by exposing unc-2 mutant animals to exogenous serotonin and dopamine, it has been found that mutations in unc-2 prevent adaptation to the presence of these neurotransmitters (Schafer and Kenyon, 1995). For example, when wild-type and unc-2 mutant 30 animals are exposed to exogenous dopamine, they both become paralyzed, but wild-type worms typically recover after several hours and begin moving and foraging. In contrast, unc-2 mutant animals remain paralyzed, implicating UNC-2 in this adaptation process (Schafer and Kenyon, 1995). unc-2 has also be found to be required for the proper neuronal migration of a subset of cells in C. elegans (Tam et al., 2000). The touch receptor neuron AVM, the motor neurons VC4 and VC5 and the interneuron SDQR migrate randomly in unc-2 mutants resulting in the misplacement of their cell bodies. However, unc-2 mutations do not affect the neuron's ability to differentiate and extend its axon in the normal pattern. Thus, UNC-2 appears to be only involved in the process of cell migration in these neurons and does not impact axon pathfinding or cell identity. In addition, UNC-2 has also been found to be involved in the transcriptional regulation of an odorant receptor encoded by the str-2 gene in the AWC olfactory neurons (Troemel et al., 1999). Under wild-type conditions a stochastic, but coordinated decision ensures that the str-2 gene is expressed in only one of the two AWC sister neurons, but never in both. Mutations to unc-2 alter this decision making process and cause str-2 to be expressed in the AWC neurons in an unregulated manner (Troemel et al., 1999). egl-19 A second Ca channel gene identified by molecular genetic approaches was the egl-19 gene which is predicted to encode the ai subunit of an L-type Ca 2 + channel (Lee et al., 1997). Behavioral analysis of egl-19 mutant animals reveals that egl-19 affects a wide variety of behaviors including egg-laying (Trent et al., 1983; Lee et al., 1997), locomotion (Lee et al., 1997; Jospin et al., 2002), pharyngeal pumping (Lee et al., 1997) and male mating (Lee et al., 1997; Garcia et al., 2001). EGL-19 is thought to function as the predominant Ca 2 + channel mediating muscle contraction (Lee et al., 1997; Jospin et al., 2002). The proper execution of egg-31 laying, locomotion, pharyngeal pumping and male mating all require the coordinated contraction of different muscle groups in C. elegans. Thus, it is reasonable to assume that mutations to the EGL-19 Ca 2 + channel involved in muscle contraction should affect behaviors requiring the proper functioning of muscles. A close phenotypic examination of different alleles of egl-19 has provided information regarding its biological roles. Loss-of-function alleles of egl-19 are embryonic lethal likely as a result of a lack of Ca 2 + channel activity necessary for body wall muscle function during development (Williams and Waterston, 1994; Lee et al., 1997; Moerman and Fire, 1997). Gain-of-function alleles of egl-19 produce myotonia and alterations to pharyngeal pumping due to a prolongation of the muscle action potential and a delay in relaxation (Lee et al, 1997). Likewise, reduction-of-function mutations cause a flaccid phenotype and alterations to pharyngeal pumping due to slow muscle depolarization and feeble contraction (Lee et al., 1997; Jospin et al., 2002). unc-36 The last of the Ca 2 + channel subunit genes identified by previous molecular genetic approaches was the unc-36 gene which is predicted to encode an a28 subunit (Schafer et al, 1996). Behavioral analyses of unc-36 mutant animals have suggested that the UNC-2 and UNC-36 Ca channel subunits may function together in vivo (Mathews et al., 2003). In support of this hypothesis, unc-36 mutant animals exhibit a number of behaviors that are very similar to that of unc-2 mutants including constitutive egg-laying, aldicarb resistance (Nguyen et al, 1995) and failure to adapt to serotonin and dopamine (Schafer and Kenyon, 1995; Schafer et al., 1996). In addition, under most behavioral assays, unc-2; unc-36 double mutants can not be distinguished from either single mutant alone further suggesting that they may function together in the same pathway (Schafer et al, 1996; Mathews et al., 2003). 32 cca-1 An additional Ca 2 + channel ai subunit gene has recently been identified by screening the C. elegans genome database (Bargmann, 1998; Cribbs et al., 1998; Perez-Reyes et al., 1998; Lee et al., 1999b; McRory et al., 2001). The putative Ca 2 + channel ai subunit gene found in cosmid C54D2.5 is predicted to encode the oti subunit of a T-type Ca 2 + channel. This gene has since been named, cca-1, and its expression has been shown to be necessary for the efficient initiation of pharyngeal muscle action potentials in response to excitation by the MC motor neuron (K. Steger and L. Avery, pers. comm.). Additional Calcium Channel Genes A second putative Ca 2 + channel a28 subunit gene encoded in cosmid T24F1.6 has been identified although its function still remains to be determined. Two (3 subunit genes have also been found in C. elegans on cosmids T28F2.5 and W10C8.1 and have been named ccb-1 and ccb-2, respectively. A loss-of-function mutation in ccb-1 generated by the C. elegans Gene Knockout Consortium has been found to be an embryonic lethal possibly due to its association with the EGL-19 Ca 2 + channel (C. Thacker, pers. comm.). A loss-of-function mutation in ccb-2 has also been isolated by the C. elegans Gene Knockout Consortium, but it displays no overt phenotype (C. Thacker, pers. comm.). All of the identified four domain-type VGIC genes in C. elegans are listed in Table 1. Two additional outlier VGIC ai subunit genes have also been identified in cosmids CI 1D2.6 and C27F2.3, named nca-1 and nca-2, respectively and are the subject of this study (Table 1). 33 Table 1. Four Domain-Type Voltage-Gate Ion Channel Genes in C. elegans Gene Protein Cosmid Phenotype Reference unc-2 Non L-type Ca 2 + channel cti subunit T02C5.5 uncoordinated, thin, egg-laying constitutive, pharyngeal pumping & defecation defects (Schafer and Kenyon, 1995) egl-19 L-type Ca 2 + channel cti subunit C48A7.1 null is embryonic lethal egg-laying, pharyngeal pumping & mating defects (Lee et al., 1997) cca-1 T-type Ca 2 + channel cti subunit C54D2.5 pharyngeal pumping & egg laying defects K. Steger, L. Avery, C. Thacker and T. Snutch, pers. comm. nca-1 NCA channel ax subunit C11D2.6 no overt phenotype this study nca-2 NCA channel cii subunit C27F2.3 no overt phenotype this study unc-36 Ca 2 + channel a28 subunit C50C3.9 uncoordinated (almost paralyzed), thin (Schafer et al., 1996) Ca 2 + channel a28 subunit T24F1.6 unknown CGC* ccb-1 Ca 2 + channel f3 subunit T28F2.5 embryonic lethal CGCVGKC" ccb-2 Ca 2 + channel 3 subunit W10C8.1 no overt phenotype CGCVGKC 1" CGC* = C. elegans Genetics Center GRC^ = C. elegans Gene Knockout Consortium 34 Sodium Channels The genome of C. elegans lacks any predicted proteins with high sequence homology to vertebrate Na + channel a subunits (Bargmann, 1998). The lack of Na + channels in the genomes of nematodes is consistent with the inability to detect any Na+-based action potentials in electrophysiological recordings made from numerous different neurons in C. elegans (Goodman et al., 1998), as well as in the motor neurons of the related nematode, Ascaris (Davis and Stretton, 1989a, b). Potassium Channels In contrast to the relatively small number of Ca 2 + channel genes present in the genome of C. elegans, there are substantially more K + channel genes. Analysis of the C. elegans genome sequence reveals that there are upwards of 80 separate K + channel genes in C. elegans (Wei et al., 1996; Bargmann, 1998). Of these predicted K + channel genes, 20 are members of the six transmembrane family of K + channels and include representatives of the five different subclasses within this family (Wei et al., 1996; Bargmann, 1998). The dramatic increase in the number of K + channel genes in C. elegans may reflect channel diversification required to modulate the electrical properties of individual neurons, thereby making them different from one another. Research Objectives: Molecular Cloning and Genetic Characterization of Novel Four Domain-Type Voltage-Gated Ion Channels While considerable progress has been made towards identifying the different types of four domain-type VGICs in vertebrates and invertebrates, there still exist a number of ionic conductances that remain to be fully characterized. Evolutionary analysis of the family of four domain-type VGICs predicts that the Ca 2 + and Na + channel families arose from an ancestral Re-type channel through two rounds of gene duplication and divergence (Hille, 1989; Strong et al., 35 1993). Based upon this proposed model of gene evolution, it is conceivable that in addition to the well characterized Ca and Na channels, there may be other families of four domain-type VGICs in vertebrates or invertebrates that are responsible for some of the uncharacterized conductances. Likewise, it is reasonable to assume that some of these conductances may be mediated by additional members of the Ca 2 + and Na + channel families that have not been identified by using classical genetic or conventional homology-based cloning techniques. The major goal of this study was to identify and characterize novel four domain-type VGIC genes. The approach taken was to screen the C. elegans genomic and GenBank EST databases using oligonucleotide sequences based upon structurally conserved regions present in the majority of mammalian HVA Ca 2 + channel a.\ subunits. Once full-length cDNAs of these newly identified VGICs were constructed, the aim was to describe their electrophysiological and pharmacological properties using standard electrophysiological analyses. Utilizing the C. elegans model system, a further aim was to construct deletion mutations in the new a\ subunit genes and examine their biological roles using a combination of genetic and behavioral approaches (see Introduction to Chapter 4). 36 Chapter 2 . Materials and Methods Screening of Genome Databases for Novel Voltage-Gated Ion Channels Identification of the nca-1, nca-2 and rat-wca Genes A homology-based strategy was utilized in order to identify novel four domain-type VGIC ai subunits. Oligonucleotide sequences were designed based upon structurally conserved regions present in five different rat HVA Ca 2 + channel ai subunits (Table 2). These oligonucleotide sequences were subsequently used to "screen" the C. elegans genome data base in early 1997 prior to the release of the entire C. elegans genome sequence (C. elegans Sequencing Consortium, 1998) and the subsequent identification of the ai subunit family of VGIC genes in C. elegans by Bargmann (1998). This screen resulted in the identification of five potential four domain-type VGIC oti subunit genes in C. elegans (cosmids and reading frames: T02C5.5, C48A7.1 C54D2.5, CI 1D2.6 and C27F2.3). Three of these ai subunit genes (T02C5.5, C48A7.1 C54D2.5) were determined to represent homologues of rat and human Ca 2 + channels, whereas the other two ai subunit genes (CI 1D2.6 and C27F2.3) appeared to represent a novel family of four domain-type VGICs. The T02C5.5 open reading frame encodes unc-2 which is a Ca 2 + channel ai subunit most similar to the mammalian CXIA, otie and ctiE non L-type channels (Schafer and Kenyon, 1995), while C48A7.1 encodes egl-19 which is a Ca 2 + channel ai subunit 37 Table 2. Oligonucleotide Sequences Used to Screen the C elegans Genome Database Primer Position(bp)/Clone(Accession #) Sequence (5' - 3') S/A 1 1370-1475/rat brain a,A(M64373) GTCAAAACTCAGGCCTTCTA S 2 2125-2148/rat brain ctlA(M64373) AACGTGTTCTTAGCTATCGCGGTG s 3 1438-1461/rat brain aiB(M92905) GTGAAAGCACAGAGCTTCTACTGG s 4 2107-2130/rat brain a1B(M92905) AACGTTTTCTTGGCCATTGCTGTG s 5 1821-1844-rat brain aiC(M67515) GTTAAGTCCAACGTCTTCTACTGG s 6 2496-2519/rat brain a,c(M67515) AATGTGTTCTTGGCCATTGCGGTG s 7 1591-1614/rat brain a1D(AF370009) GTGAAGTCTGTCACGTTTTACTGG s 8 2266-2289/rat brain aiD(AF370009) AAGCTCTTCTTGGCCATTGCTGTA s 9 1464-1484/rat brain a,E(L15453) GTCAAGTCGCAAGTGTTCTAC s 10 2133-2152/rat brain a1E(L15453) AATGTATTCTTGGCTATCGC s S/A = sense/antisense primer 38 homologous to the mammalian otic, am, aiF and ais L-type channels (Lee et al., 1997) and C54D2.5 encodes cca-1 which is a Ca 2 + channel ai subunit homologous to the mammalian am, am and an T-type channels (Bargmann, 1998; Cribbs et al., 1998; Perez-Reyes et al., 1998; Lee et al., 1999b; McRory et al., 2001). I have since named CI 1D2.6 and C27F2.3, nca-1 and nca-2, respectively for "novel four domain-type VGIC cti subunit". Mammalian homologues of nca-1 and nca-2 were identified by screening the GenBank EST and non-redundant data banks with the nca-1 and nca-2 gene sequences. Four resulting ESTs (AA157945, AA683293, AA967995 and AF078779) were judged to encode novel NCA-like proteins. Genetic Analyses Nematode Strains and Growth Conditions All C. elegans strains were grown on nematode growth media (NGM) plates streaked with Escherichia coli (strain OP50) and incubated at 20°C (Brenner, 1974; Lewis and Fleming, 1995). All techniques and solutions used in the propagation and maintenance of the strains are outlined in "Basic Culture Methods. In Methods in Cell Biology: Caenorhabditis elegans: Modern Biological Analysis of an Organism" by J. Lewis and J. Flemming (1995). Some strains were provided by E. M Jorgensen (University of Utah, Salt Lake City, UT), D. G. Moerman (University of British Columbia, Vancouver, Canada) and J. A. Dent (McGill University, Montreal, Canada). Additional strains were provided by the C. elegans Genetics Center (CGC) and the C. elegans Gene Knockout Consortium. Strains in this work include the wild-type strain N2, CB129 unc-2 (el29)X, CB156 unc-25(el56)III, EG8 unc-2(ox8)X, JD21 cca-1 (adl650)X, MT8189 lin-15(n765ts)X, TS4 vaEx3, TS46 nca-2(gk5)IU, TS48 vals6, TS61 nca-1(gk9)IV, TS62 cca-1 (adl650)X; nca-l(gk9)IV, 39 TS64 cca-1 (adl650)X; nca-2(gk5)III, TS65 nca-2(gk5)M; nca-1(gk9)IV, TS105 cca-l(adl650)X, TS124 unc-2(ox8)X, TS144 unc-2(ox8) cca-1 (ad!650)X, TS164 valsll, TS169 unc-2(e!29)X, TS174 vaEx!8, TS191 va/s75, TS199 valsll; vals!5, TS214 unc-2(el29)X; nca-2(gk5)III; nca-1(gk9)IV, TS215 unc-2(el29)X; nca-2(gk5)III, TS216 unc-2(el29)X; nca-l(gk9)IV, TS217 unc-2(ox8)X; nca-2(gk5)III; nca-1(gk9)IV, TS218 wnc-2(ax8)X; nca-2(gk5)III, TS241 cca-1 (adl650)X; nca-2(gk5)III; nca-1 (gk9)IV, TS242 «rtc-2foxc?;.A'; nca-l(gk9)IV, TS243 unc-2(ox8) cca-1 (adl650)X; nca-2(gk5)IU; nca-1(gk9)IV, TS245 unc-25(el 56)111; nca-l(gk9)IV, TS246 nca-2(gk5) unc-25(el 56)111; nca-1 (gk9)IV, TS259 nca-2(gk5) unc-25(el 56)111 VC9 nca-2(gk5)III, VC12 nca-1 (gk9)IV. Isolation of VC12 nca-1 (gk9)IV and VC9 nca-2(gk5)III Deletion Mutant Strains As nca-7 and nca-2 were first identified by "screening" the C. elegans genome data base, no visible mutations had previously been reported for either gene. A recently developed PCR-based gene knockout approach was used to isolate putative null alleles of nca-1 and nca-2 (Jansen et al., 1997; Liu et al., 1999). These two alleles, nca-1(gk9) and nca-2(gk5), were generated by the C. elegans Gene Knockout Consortium at the University of British Columbia. Molecular analysis of the VC12 nca-l(gk9)IV knockout strain indicated that the molecular lesion in nca-1(gk9) is a 2287 bp deletion that spans from bp 15464 to bp 17750 on cosmid CI 1D2 (AF045640) and there is also a 128 bp insertion at the deletion breakpoint. The deficiency spans from the end of intron 13 to the middle of intron 18 of the nca-1 gene (Figure 6A). Similarly, molecular analysis of the VC9 nca-2(gk5) knockout strain indicated that the molecular lesion in nca-2(gk5) is a 2970 bp deletion that spans from bp 16483 to bp 19452 on cosmid C27F2 (U40419). The deficiency spans from the end of intron 7 to the beginning of exon 16 of the nca-2 gene (Figure 6B). 40 Figure 6. Diagrams Illustrating the PCR Strategies Used to Identify Wild-type Versus Deletion Alleles for nca-1, nca-2 and cca-1 For all gene diagrams, exons and introns are represented as boxes and lines respectively. Primer sets are indicated on the gene diagrams as arrows and the arrowhead indicates the approximate location of the primer. The region of the gene deleted in the nca-1 (gk9), nca-2(gk5) and cca-1 (ad1650) alleles is indicated with a red box. A) The molecular lesion in the nca-1 (gk9) allele is a 2287 bp deletion with a 128 bp insertion at the deletion breakpoint. PCR using the primer set KH4-KH6 (located within the deletion) was used to detect the wild-type allele and amplified a 660 bp product. PCR using the primer set NliL-NliR (flanking the deletion breakpoint) was used to detect the gk9 allele and amplified a 837 bp product (amplification from wild-type (2996 bp) was selected against by using a short PCR extension time). B) The molecular lesion in the nca-2(gk5) allele is a 2970 bp deletion. PCR using the primer set KH102A-KH102S (located within the deletion) was used to detect the wild-type allele and amplified a 292 bp product. PCR using the primer set N2iL-N2iR (flanking the deletion breakpoint) was used to detect the gk5 allele and amplified a 435 bp product (amplification from wild-type (3405 bp) was selected against by using a short PCR extension time). C) The molecular lesion in the cca-1 (adl650) allele is a 2405 bp deletion. PCR using the primer set cca29-IR2 (located within the deletion) was used to detect the wild-type allele and amplified a 1160 bp product. PCR using the primer set cca26-cca27 (flanking the deletion breakpoint) was used to detect the adl650 allele and amplified a 957 bp product (amplification from wild-type (3362 bp) was selected against by using a short PCR extension time). 41 42 In order to reduce the likelihood of any additional mutations elsewhere in the genomes of the two stains, they were each backcrossed six times with N2 males. Neither VC12 nor VC9 showed any overt morphological phenotypes, therefore during backcrossing a PCR-based approach was used to follow both the gk.9 and gk5 deletion alleles and determine the genotype of progeny hermaphrodites. This PCR-based approach took advantage of the fact that the molecular lesions in both VC12 and VC9 are large deletions. The VC12 nca-1(gk.9) mutant strain has a 2287 bp deletion with a 128 bp insertion at the deletion breakpoint and the VC9 nca-2(gk5)III mutant strain has a 2970 bp deletion. Consequently, PCR reactions that use primer pairs homologous to regions within the deletion should amplify a PCR product when the wild-type allele is present. Similarly, PCR reactions that use primer pairs quite close to the deletion breakpoints should amplify a PCR product when the deletion allele is present if the PCR reaction extension time is biased towards amplification of the short deletion product (Figure 6). To backcross the VC12 nca-1 (gk9)IV strain, N2 males were crossed to VC12 nca-1 (gk9)IV hermaphrodites. Backcrossed hermaphrodites (gk9/+) were then allowed to self-fertilize. Twelve of the progeny hermaphrodites (+/+ : 2 gk9/+ : gk9lgk9) were placed onto separate plates and allowed to self-fertilize and lay eggs. After approximately 24 hrs, genomic DNA was isolated from each worm (described below) and tested using PCR for the presence of the gk.9 deletion allele. To detect the gk9 deletion allele, primers flanking the deletion breakpoint (NliL-NliR) were used to amplify a 837 bp deletion product. The corresponding 2996 bp wild-type PCR product was biased against using a short extension time. Similarly, to detect the wild-type allele, primers within the deletion (KH4-KH6) were used to amplify a 660 bp wild-type product (Figure 6A). Lines that only amplified the 837 bp gk9 deletion allele (gk9/gk9) were kept for further backcrossing. The above strategy was repeated an additional five times with N2 males being crossed to the newly isolated hermaphrodite line (gk9/gk9) from the previous round of 43 backcrossing. After backcrossing a total of six times, the VC12 strain was named TS61 nca-l(gk9)IV. Similarly, in order to backcross the VC9 nca-2(gk5)IH strain, N2 males were crossed to VC9 nca-2(gk5)III hermaphrodites. Backcrossed hermaphrodites (gk5/+) were then allowed to self-fertilize. Twelve of the progeny hermaphrodites (+/+ : 2 gk5/+ : gk5lgk5) were placed onto separate plates and allowed to self-fertilize and lay eggs. As described above, after approximately 24 hrs, genomic DNA was isolated from each worm (described below) and tested using PCR for the presence of gk5 deletion allele. To detect the gk5 deletion allele, primers flanking the deletion breakpoint (N2iL-N2iR) were used to amplify a 435 bp deletion product. The corresponding 3405 bp wild-type PCR product was biased against using a short extension time. Similarly, to detect the wild-type allele, primers within the deletion (KH102A-KH102S) were used to amplify a 292 bp wild-type product (Figure 6B). Lines that only amplified the 435 bp gk5 deletion allele (gk5lgk5) were kept for further backcrossing. The above strategy was repeated an additional five times with N2 males being crossed to the newly isolated hermaphrodite line (gk5lgk5) from the previous round of backcrossing. After backcrossing a total of six times, the VC9 strain was named TS46 nca-2(gk5)III. Construction of the TS65 nca-2(gk5)III; nca-1 (gk9)IV Double Mutant Strain To construct the nca-2(gk5); nca-1 (gk.9) double mutant, N2 males were crossed to TS46 nca-2(gk5)III hermaphrodites. Outcrossed males (gk5/+) were mated to TS61 nca-1 (gk9)IV hermaphrodites and the genotype of the hermaphrodite progeny was determined by PCR (described above). Ten of the hermaphrodites (gk5/+ ; +/gk9 or +/+ ; +/gk9) were placed onto separate plates and allowed to self-fertilize and lay eggs. After approximately 24 hrs, each worm was tested using PCR for the presence of gk5 deletion allele. Only lines that amplified the gk5 deletion allele (gk5l+ ; +/gk9) were kept for further analysis. From one of these lines, 30 44 hermaphrodite progeny were picked onto separate plates, allowed to self-fertilize and lay eggs. Selected hermaphrodites were then tested using PCR for both the wild-type and deletion alleles at both the nca-2 and nca-1 loci. The line that did not amplify either of the wild-type alleles, but only amplified both the gk5 and gk9 deletion alleles (gk5/gk5 ; gfc9/gk9) was selected as the source of the double mutant strain, TS65 nca-2(gk5)HI; nca-1 (gk9)IV. Construction of cca-1; nca Mutant Strains To construct the different cca-1; nca mutant strains, classical genetic techniques and the PCR strategies outlined above were utilized. In addition, a PCR strategy was also used to follow the adl 650 deletion allele. This PCR strategy took advantage of the fact that the molecular lesion of the adl650 allele is a 2405 bp deletion. Therefore, PCR primer sets were designed within the deletion (primer set cca29-IR2 amplifying a 1160 bp wild-type product) and just flanking the deletion breakpoint (primer set cca26-cca27 amplifying a 957 bp deletion product). The corresponding 3362 bp wild-type PCR product was biased against using a short extension time (Figure 6C). To construct these mutant strains, N2 males were crossed to JD21 cca-1(adl 650JX or TS105 cca-1 (adl650)X hermaphrodites and outcrossed males were mated to hermaphrodites from the three different nca mutant strains. Thirty F3 hermaphrodites were tested using PCR for the adl650, gk5 and/or gk9 deletion alleles. Positive worms were picked and propagated until they were homozygous for the adl650 and gk5 and/or gk9 deletion alleles. From this approach, the strains TS62 cca-1(adl650JX; nca-1(gk9)IV, TS64 cca-1 (adl650JX; nca-2(gk5)lll and TS241 cca-1(adl 650JX; nca-2(gk5)III; nca-1(gk9)IV were constructed. Construction of unc-2; nca Mutant Strains To construct the different unc-2; nca mutants, classical genetic techniques and the PCR strategies outlined above were utilized. Two different strains of unc-2 were used, EG8 unc-45 2(ox8)X and CB129 unc-2 (el29)X renamed TS124 unc-2(ox8)X and TS169 unc-2(el29)X, respectively after backcrossing. Briefly, N2 males were crossed to TS65 nca-2(gk5)IH; nca-1 (gk9)IV hermaphrodites and outcrossed males were mated to the appropriate Unc-2 hermaphrodites (TS124 or TS169). Eight F 2 hermaphrodites were picked onto separate plates and were tested using PCR for the presence of both the gk5 and gk9 deletion alleles. Only lines that had both deletion alleles were kept. Between 10 and 32 F3 Unc-2 hermaphrodites were picked and their genotype at the nca-1 and/or nca-2 loci was determined by PCR. Positive worms were picked and propagated until the gk5 and/or gk9 alleles were driven to homozygousity. From this strategy, the strains TS214 unc-2(e!29)X; nca-2(gk5)III; nca-l(gk9)IV, TS215 unc-2(el29)X; nca-2(gk5)III, TS216 unc-2(el29)X; nca-l(gk9)IV, TS217 unc-2(ox8)X; nca-2(gk5)III; nca-1 (gk9)IV, TS218 unc-2(ox8)X; nca-2(gk5)III and TS242 unc-2(ox8)X; nca-1(gk9)IV were constructed. Construction of unc-2(ox8) cca-1(adl650); nca Mutant Strains The strategy utilized to construct the unc-2(ox8) cca-1 (ad1650); nca-2(gk5); nca-1(gk.9) mutant strain was similar to that used to construct the unc-2(0x8); nca mutant strains except that in addition to testing for the gk5 and/or gk9 deletion alleles by PCR, the adl650 deletion allele was always tested to ensure that it had not been lost due to homologous recombination during outcrossing. The parent strain used was TS144 unc-2(0x8) cca-1 (adl650)X and the strain constructed as a result of this strategy was TS243 unc-2(0x8) cca-1 (adl650)X; nca-2(gk5)III; nca-1 (gk9)IV. Construction Of unc-25; nca Mutant Strains To construct the different unc-25; nca mutants, classical genetic techniques and the PCR strategies outlined above were utilized. Briefly, N2 males were crossed to CB156 unc-46 25(el56)111 hermaphrodites and outcrossed males were mated to the appropriate hermaphrodites (TS46 nca-2(gk5)III, TS61 nca-1(gk9)IV or TS65 nca-2(gkSJIII; nca-1 (gk9)IV). Eight F 2 hermaphrodites were picked onto separate plates and scored for their ability to segregate the Unc-25 phenotype. Only lines that segregated Unc-25 were kept. Between 32 and 48 F 3 Unc-25 hermaphrodites were picked and their genotype at the nca-1 and/or nca-2 loci was determined by PCR. Positive worms were picked and propagated until the gk5 and/or gk9 alleles were driven to homozygousity. From this strategy, the strains TS245 unc-25 (el56)111; nca-1 (gk9)IV, TS246 nca-2(gk5) unc-25(el 56)111; nca-1 (gk9)IV and TS259 nca-2(gk5) unc-25 (el56)111were constructed. Construction of the TS199 valsll; valslS Transgenic Strain To construct the TS199 valsll; valslS transgenic strain classical genetic techniques were used. GFP expressing TS 191 valslS hermaphrodites (containing the integrated pnca-2::GFP reporter construct) were mated to N2 males. GFP expressing Fi outcrossed males (valsl 5/+) were mated to DsRed2 expressing TS164 valsll hermaphrodites (containing the integrated p«ca-/::DsRed2 reporter construct). The resulting F 2 hermaphrodites (valsll'/+; vals!5/+ or valsll/+; +/+) were examined for GFP expression, which indicated the presence of the valslS allele. Two GFP/DsRed2 positive F 2 hermaphrodites (valsll/+; valsl5l+) were placed onto separate plates and allowed to self-fertilize. The selection of candidate F 3 hermaphrodites for further propagation was made easier because the intensity of DsRed2 fluorescence emitted from hermaphrodites homozygous at the valsll locus (valsll/valsl 1) were brighter than those that were heterozygous at that locus (valsl 1/+). Twelve F 3 hermaphrodites that had bright DsRed2 expression, as well as GFP fluorescence (valsll/valsll; valsl5/+ or valsl 1/valsll; valsl 5/vaIs 15) were placed onto separate plates and allowed to self-fertilize. Only F 4 hermaphrodites (valsl 1/valsll; valsl5/valsl5) that segregated GFP and DsRed2 expressing 47 offspring were kept and were propagated for a number of generations to ensure homozygousity at both loci. DNA Transformation and Microinjection of C. elesans The DNA transformation techniques used in this study are as outlined in "DNA Transformation. In Methods in Cell Biology: Caenorhabditis elegans: Modern Biological Analysis of an Organism" by C. Mellos and A. Fire (1995). MT8189 /m-75fn7c)5r5JXhermaphrodites were chosen as the background strain to be injected so that the co-injection marker plin-15(+) could be used to identify transformants. lin-15 encodes for two novel proteins that function in the hypodermis during vulval formation. One mutant allele of this gene, Un-15(n765ts), results in a multivulva (Muv) phenotype when propagated at 20°C but is wild-type (non-Muv) at 15°C. This Muv phenotype can thus be rescued by co-injection of the plin-15(+) plasmid, which restores a wild-type copy of the lin-15 gene (Clark etal., 1994). DNA used in the injection cocktail was generated using standard molecular biology techniques (Sambrook and Russell, 2001). The injection cocktail consisted of 50 ng/ul reporter construct of interest, 50 ng/ul of the co-injection marker plasmid plin-15EK (Clark et al., 1994) and 25 ng/ul pBluescript SK- (Stratagene) used as carrier DNA. The injection cocktail was centrifuged at maximum speed in a microfuge for 15 min prior to use to ensure that there were no particulate contaminants that would clog the injection needle. Microinjection needles were constructed from borosilicate glass tubing (Sutter Instrument Co.) on a P-87 micropipette puller. Needles were loaded with injection cocktail and between 20 and 40 well-feed MT8189 lin-15(n765ts)Xyoung adult hermaphrodites were mounted onto 2% agarose pads and injected with the DNA cocktail into their syncytial gonads (Mello and Fire, 1995). The microinjection 48 experiments were performed by Dr. C. Thacker (T. Snutch Laboratory, University of British Columbia). Identification of Transformants and Establishing Transgenic Lines After injection, Lin-15 worms were recovered by placing the worm in a drop of M9 buffer (g/1: 6 NaHP04, 3 KH 2P0 4 , 5 NaCI, 0.25 MgS0 4 7H20) and transferring them individually to a freshly seeded NGM plate. The injected worms were allowed to self-fertilize and lay eggs at 20°C and single wild-type (non-Muv) progeny from each injected Lin-15 worm were picked onto separate plates. Each of the wild-type hermaphrodite lines were allowed to self-fertilize at 20°C and were inspected for their ability to propagate the extrachromosomal array. Only those lines that had more than 50% wild-type (non-Muv) progeny and expressed the reporter construct of interest were kept for further propagation. All putative lines were examined over several generations to ensure that the extrachromosomal array was stably propagated to the next generation. From this approach the transgenic strains TS4 vaEx3 and TS174 vaEx!8 containing extrachromosomal arrays were generated. Integration of Extrachromosomal Arrays Fifty well-fed wild-type (non-Muv) L4 hermaphrodites containing the extrachromosomal array of interest were transferred to an NGM plate seeded with a spot of OP50. The plated was then irradiated with 2500 rad of gamma rays from a 6 0Co source for 25 min. Each of the irradiated hermaphrodites were transferred to separate plates and allowed to self-fertilize and lay eggs for approximately 12 hrs at 20°C (plates 1A to 50A). Worms were subsequently transferred to fresh plates every 12 hrs for a total of three transfers (plates B, C and D for a total of 200 plates). All plates were then allowed to self-fertilize at 20°C for at least two generations (until F2s appeared), starved and stored at 15°C for later use. Plates were then chunked out in groups of 49 10 starting with the B plates (IB to 10B) and allowed to recover at 20°C. From each recovered plate, 15 wild-type hermaphrodites were picked at random onto separate plates for all of the ten starved plates chunked (1B-1 to 1B-15... 10B-1 to 10B-15). All 150 plates were then allowed to self-fertilize and subsequently scored for the presence of 100% wild-type (non-Muv) progeny. Plates with any Lin-15 (Muv) hermaphrodites were discarded. Plates that only segregated wild-type (non-Muv) worms were used to establish putative integrated lines and were followed for several generations to ensure that they only segregated wild-type (non-Muv) hermaphrodites and expressed the reporter construct of interest. At most, only one line per original mutagenized hermaphrodite was kept and used as the source strain for the newly integrated lines. If no lines were obtained after screening of the first 150 plates, the process was repeated with as many of the original starved plates as needed. From this approach the transgenic strains TS 48 vals6, TS164 valsll and TS191 valsl5 containing integrated arrays were generated. Fluorescent Microscopy of C. elesans In order to determine the cellular expression pattern of nca-1 and nca-2, transgenic hermaphrodites that contained the GFP (and DsRed2 in the case of TS199) reporter construct(s) of interest as a stably integrated array were examined. Transgenic hermaphrodite worms were placed in a drop of 0.3 M BDM (Sigma) in M9 buffer and mounted on 2% agarose pads under a 1 mm glass slide. A ring of Vaseline was used to seal the specimen and prevent sample desiccation. Cell identification was determined based upon the position and characteristic morphology of GFP-positive nuclei using a combination of fluorescent and Nomarski differential interference contrast (DIC) microscopy. 50 Phenotypic Analysis of nca Mutants Aldicarb and Nicotine Resistance NGM plates were prepared according to standard methods. Aldicarb (Sigma) was prepared as a 100 mM stock in 70% ethanol and added to NGM media solution to a final concentration of 1 mM (Miller et al., 1996). Assay plates were seeded with a small spot of OP50 and kept in the dark at 4°C until used. All strains were tested on the same batch of assay plates. For each assay, 20 well-fed adult worms were placed onto the spot of OP50 and were tested for paralysis at 10 min intervals for a total of 2 hrs. Paralysis was defined as the lack of spontaneous movement, an inability to move when the plate was tapped and an inability to move when prodded with a pick. All scoring was performed blind to genotype. The data was fitted with the equation: y = min + (max - min)/(l + [x/x50]k) where: y = % of animals paralyzed min = the minimal % of animals paralyzed (set at zero, because the assay started with all animals able to move) max = the average of the maximum % of animals paralyzed for that strain x = the time the animals had been on 1 mM aldicarb x 5 0 = the time at which 50% of the animals were paralyzed k = the slope of the curve Significant resistance to aldicarb was determined by ascertaining whether the onset of paralysis curve for a given mutant strain on aldicarb was significantly different (P < 0.05) from that for wild-type using the program Allfit (Waud, 1972; van Swinderen et al., 1997). 51 To test for sensitivity to nicotine, plates containing a population of worms were flooded with a 1% solution of nicotine (Sigma). After 10 min, worms were scored for hypercontraction (Brenner, 1974; Lewis et al, 1980). Halothane Assay The effect of the volatile anesthetic, halothane was quantified by the radial dispersal assay. Well-feed young adult worms were washed off of 3-4 NGM plates with 1 ml of S-Basal (5.9 g/1 NaCI, 50 mM KH 2P0 4 , 5 mg/1 cholesterol) and placed into a microfuge tube. After the worms were allowed to settle to the bottom of the tube, the supernatant was pulled off and another ml of S-Basal was added. After a third wash, the worms were rinsed once with dH20 and resuspended in 100 ul of dH20. 10 u.1 aliquots containing 50-100 worms were placed onto the center of the dispersal assay plate (NGM plates seeded with a ring of OP50 along the outer edge of the plate). Halothane (Halocarbon Products) was injected as a liquid into air-tight glass chambers (Corning) that contained the assay plates for each strain. Once the water had soaked in, the chamber was shaken until the worms began dispersing and timing began. After 40 min, the number of worms that were in the bacteria ring, as well as the number of worms that were not in the ring was counted. From this data, the radial dispersion index was calculated as the number of worms in the ring of bacteria after 40 min divided by the total number of worms on the plate. After the experiment, gas phase halothane concentrations were measured by gas chromatography and the volume percentage (vol%) determined by interpolation against standards. This process was repeated for a range of different halothane concentrations (between 0 and 2.5 volume percent). The EC50 (the effective halothane concentration for a 50% reduction in the radial dispersal index) was calculated from the plot of radial dispersal index verses halothane concentration (Crowder et al., 1996). 52 The data was fitted with the equation: y = min + (max - min)/(l + [x/x50]"k) where: y = radial dispersal index min = the minimal radial dispersal index (assumed to be zero, because all curves approached zero) max = the average of the maximum radial dispersal indices for that strain x = the concentration of halothane x 5 0 = the E C 5 0 k = the slope of the curve Significant hypersensitivity of a strain's EC50 was determined by ascertaining whether the halothane dose-response curve for a given mutant strain was significantly different (P < 0.05) from that for wild-type using the program Allfit (Waud, 1972; van Swinderen et al., 1997). The halothane assay was performed by L. Metz (M. Crowder Laboratory, University of Washington, St. Louis). Body Bends Assay Well-fed young adult worms were picked onto unseeded NGM plates and allowed to crawl free of any adherent bacteria for 1 min. Worms were then picked onto a second unseeded NGM plate that was pre-equilibrated to 20°C and were allowed to recover for 1 min. After recovery, the worm was scored by eye for the number of body bends that occurred in three consecutive 30 s intervals. All of the body bend assays were conducted in a temperature controlled room set at 20°C. A body bend was defined as a change in direction of bending at the mid-body and was scored blindly with regards to genotype (Robatzek and Thomas, 2000; Tsalik 53 and Hobert, 2003). Ten animals from each strain were examined and significant differences (P < 0.05) between the observed rates of body bends were determined by One-way ANOVA (Origin). Thrashing Assay Individual well-fed L4 hermaphrodites were picked off of their colony plates and transferred to a chambered petri plate containing 150 ul of M9 buffer pre-equilibrated to 20°C. Each worm was allowed to recover for 2 min and then was videotaped for 1 min. All thrashing assays were conducted in a temperature controlled room set at 20°C. The number of thrashes per minute of each worm was analyzed by eye using stop-frame video analysis by two independent observers. A thrash was defined as a change in direction of bending at the mid-body and was scored blindly with regards to genotype (Miller et al., 1996; Tsalik and Hobert, 2003). Between nine and 20 animals from each strain were examined and significant differences (P < 0.05) between the observed rates of thrashing were determined by One-way ANOVA (Origin). Defecation Assay Well-fed young adult hermaphrodites were placed onto NGM plates seeded with a 24 hr lawn of OP50 pre-equilibrated to 20°C. Hermaphrodites were examined for the presence or absence of an expulsion event after each posterior body muscle contraction (pBoc) of the defecation cycle. Expulsion events (Ernes) are visible as a quick posterior body movement followed by the ejection of fecal material. In this study, expulsion events that consisted of posterior movement but no expulsion of fecal material were considered incomplete and were counted as failures. The time between successive pBocs was determined using Java Runtime Environment (Thomas, 1990; Miller et al, 1996). All of the defecation assays were conducted in a temperature controlled room set at 20°C. Between nine and ten animals from each strain were observed for ten consecutive cycles and all worms were scored blind to genotype. Significant 54 differences (P < 0.05) between the expulsion failure rates and differences in times between successive pBocs were determined by One-way ANOVA (Origin). Molecular Biology Standard molecular biology techniques were used (Sambrook and Russell, 2001). Subcloning of genomic sequences and cDNAs was done using the pBluescript KS and SK (Stratagene), pSL 1180 (Pharmacia), pCDNA3 and pCDNA3.1 zeo (Invitrogen), pGEM-T Easy (Promega), pPD95.67 (A. Fire), pDsRed2 (Clontech) and pCX-TOPO (Invitrogen) vectors. Ligation reactions were transformed into Epicurian coli XL-I (Blue) electrocompetent cells and bacterial colonies were grown on LB plates containing 15 g agar/1 and antibiotic at the appropriate concentration. Whenever possible, bacterial colonies transformed with recombinant plasmids were identified through blue-white selection. All of the primers mentioned in this study are listed in Table 3. The DNA sequence of the constructs were determined by the dideoxy method using the Sequenase 2.0 kit (U.S. Biochemical Corporation) or by the Nucleic Acids Protein Services Unit at the University of British Columbia. Isolation of C. elegans Genomic DNA for Genotype Testing Single worms were picked from a plate and transferred into a PCR tube containing 5 al of lysis buffer (IX PCR buffer, 1.5 mM MgCl 2 and 60 ug/ml Proteinase K). The tubes were then placed at -80°C for 15 min after which time 30 ul of mineral oil was added and the contents of the tube were collected by centrifugation. The tubes were then placed at 57°C for 60 min, then at 90°C for 15 min after which time the tubes were placed on ice for 5 min and the contents of the tube were collected by centrifugation. 1 ul of the lysed worm solution was then used in a PCR reaction. 55 Table 3. Sequence of Oligonucleotide Primers Primer Position( bp)/Clone( Accession #) Sequence (5' - 3') S/A AAP 5' RACE System (Invitrogen) G<}CCACGCGTCGACTAGTACGGGIIGGGIIGGGnG S AUAP 5' RACE System (Invitrogen) GGCCACGCGTCGACTAGTAC s cca26 31325-31306/C54D2(U37548) AGAGGACCAAGTTTACGATG s cca27 27966-27985/C54D2(U37548) GCGAGACCATTACAGAGAAC A cca29 29423-29443/C54D2(U3 7548) AGAGCCTGTTTTAGCTCTTGA A Flag A Modified FLAG epitope CTTGTCATCGTCGTCCTTGTAGTC A FlagS Modified FLAG epitope GACTACAAGGACGACGATGACAAG s IR2 30583-30564/C54D2(U37548) CTTTGACACGTTGCTCTGGG s JM91 604-587/rat a-tubulin (V01227) CCCCCCAGGTTTCCACTG s JM92 792-809/rat a-tubulin (V01227) AGGGCCCCATCAAATCTG A JM93 721-738/rat a-tubulin (V01227) ATTGAGCGCCCAACCTAC s KH4 16738-16755/C11D2(AF045640) GCCGTATATTGATTGGGC s KH6 16096-16111/C11D2(AF045640) CATGACTCCTCCGACATC A KH14 3168-3188/rat-nca A(AY555273) TTTGCAAGCTTTGGTGTTCAG s KH15 3508-3528/rat-nca A(AY555273) AAAGAGTCAGTCCAATCATGC A KH16 2883-2903/rat-»ca A(AY555273) AAGATTATGGCAGATGGCTTG S KH47A 200-2 n/rat-nca A(AY555273) ATGGCACAGATGCGCAGC A KH50A 959-976/rat-nca A(AY555273) AAGTAGGAACGCCAACGG A KH61 4785-4802/rat-raca A(AY555273) CAGACTATTCGCATGTGG S KH62 5042-5059/rat-nca A(AY555273) GGATGCAGCAGATACTGG S KH73 67-81/rat-wca A(AY555273) GAGCGGCCGCCTAACTTCACCATGC S KH79 26928-26945/C11D2(AF045640) CTGCATGCACTGACTTCCGGAGTGCC S KH790S 27089-27106/C11D2(AF045640) TTGCTCCGTCGACATCCG S KH80 77-94/«ca-/(AY555271) 23446-23463/C11D2(AF045640) GCTCTAGAATGGCTGCAATGCTCTCC A KH80OS 126-138/nca-7(AY555271) 23397-23414/C11D2(AF045640) CTCACTGCTCCTGACTGG A KH81 10849-10866/C27F2(U40419) CTGCAAGCTTGTTGTACAAGGGACCG S KH810S 10680-10697/C27F2(U40419) CTACAGCTCGTAACCTCC S KH82A 14331-14349/C27F2(U40419) CCTGCAGGATCGAAGTGCTCCAAGCC A KH82AOS 836-853/A5' »ca-2(AY555272) 14616-14633/C27F2(U40419) CAGTTCCATTGACAACGC A KH83 5255-5272/rat-«ca A(AY555273) CCACTCTCGTCACTCTCC A KH84 5191-5208/rat-wca A(AY555273) AAGCTGTGTCTGGCATGG A KH85 112-129/rat-wcar A(AY555273) AGTCAGTTACTGGCTGGG A KH87 4745-4762//ica-7 (AY555271) CCTTCCAGCCATGTTCTG A KH89 1059-1076/rat-nca A(AY555273) ATCAGAGTACAATTTCAA S KH90 1206-1223/rat-//ca A(AY555273) CCGCATCATTTTCTGTAG A KH91 4864-4881/C27F2(U40419) TCAGCGAATCCAGGTCCG S KH92 4896-4913/C27F2(U40419) TACTCCAGTTCCAGCACC S KH93 11026-11043/C27F2(U40419) CACGAGGACTCTAATTGG A KH94 11178-11195/C27F2(U40419) GCTTCAGACTCTTACTCG A KH98 6655-6672/C27F2(U40419) AATCGACACTTGGTCTGC S 5 6 KH99 6876-6893/C27F2(U40419) TAAATGAGGCTCTACTGG A KH100 9339-9356/C27F2(U40419) GTTTGATCTCCTGGCAGG S KH101 9491-9508/C27F2OJ40419) TCTTGAGCAGGGTTACGG A KH102A 19300-19317/C27F2(U40419) ACTTACAGCTCCATGTCG A KH102S 19026-19043/C27F2(U40419) AGTTCAACTAGTCGGTGG S KH104 19-38/nca-7(AY555271) CGATGATGAGTTCTTACGC A KH106 l-18/nco-/(AY555271) TGTCGTCTGCGATGCTGG S KH107 2012-2029/«ca-7(AY555271) CAGTGACTTTCCAATGCC s KH108 3340-3357/wca-/(AY555271) ATGGTCCATGTCTACTCC A KH115 309-326/A5' nca-2(AY555272) TCTCCGAGATATGGTTCG S KH116 2212-2231/A5' nca-2 (AY555272) GTCTCTACGAATTCATCCGC A KH117 858-877/«ca-/(AY555271) ACAGTGAACACACTTGCTCC A KH119 2564-2590/«car-/(AY555271) AAAGAAAAGATTCAAAGAAATTCGAGC S KH120 2564-2590/«ca-/(AY555271) GCTCGAATTTCTTTGAATCTTTTCTTT A M13R Common MCS primer CAGGAAACAGCTATGAC A NliL 14970-14989/C11D2(AF045640) CTTCCAGGCGAGTTACGAAG A NliR 17946-17965/C11 D2(AF045640) TTGTCCCTGTCGATTTGTGA S N2iL 16242-16261/C27F2OJ40419) AAATCTTTGGACCCGGAAAG S N2iR 19627-19646/C27F2(U40419) AACGCAGTTTTGCAGATTCC A SL1 Modified SL1 sequence ATAAGAATGCGGCCGCGGTTTAATTACCCAGTTTG S Bold letters = sequence not homologous to indicated clone Red letters = sequence not found in clone nca-1 (AY555271) S/A = sense/antisense primer 57 R N A Extraction from C. elegans N2 hermaphrodites were grown until crowded on a 150 mm x 15 mm NGM plate seeded with E. coli (strain C600). Worms were washed off of the culture plate with S-basal and inoculated into 400 ml of SM media (11 S-basal; 10 ml 1 M potassium citrate pH 6,10 ml trace metals solution, 3 ml 1 M CaC^, 3 ml 1 M MgSCXj) containing a 16 g pellet of C600. The culture was then incubated at 20°C for 5 days with shaking. The culture was then placed on ice for 15 min to allow the worms to settle and the culture media was removed by aspiration. The worms were then transferred to a 50 ml tube, washed once with M9 buffer and cleaned from residual bacteria by floating on a 40% sucrose solution (spun each time at 600 x g for 5 min). The worms were then washed two times with dFkO and once with DEPC-dE^O (spun each time at 800 x g for 5 min). The worm pellet was then split into two 50 ml tubes and flash frozen in liquid nitrogen. Only one of the tubes was used for the RNA extraction process. The frozen pellet was ground using a mortar and pestle under liquid nitrogen and the disrupted worms were weighed (approximately 5 g) and split into two 50 ml tubes (approximately 2.5 g/tube). Twenty-five ml of Trizol (Invitrogen) was added to each of the two tubes and they were mixed for 10 min at room temperature. Five ml of chloroform solution (0.2 ml chloroform/ml of Trizol) was added to each tube and the samples were shaken by hand for 15 s and then incubated at room temperature for 15 min. Each tube was then split into three 30 ml corex tubes and then centrifuged at 12,000 x g for 15 min at 4°C. The aqueous layer was collected and transferred to clean corex tubes. 500 ul of isopropyl alcohol was added per ml of Trizol used and the tubes were incubated at room temperature for 10 min. The tubes were subsequently centrifuged at 12,000 x g for 10 min at 4°C and the RNA pellet was washed with 50 ml of 75% EtOH made with.DEPC-dH20. Samples were mixed by vortexing and centrifuged at 7,000 x g at 4°C for 5 min. The pellet was then allowed to dry for 5-10 min and resuspended in DEPC-cfflbO. The RNA samples were then incubated at 55°C for 10 min to ensure that the RNA was fully in solution. The RNA concentration was 58 determined by spectrophotometery (A260) and the integrity of the sample was determined by gel electrophoresis. RNA Extraction from Rat Tissues One adult male and female rat were sacrificed using chlorohydrate (150 mg/kg) and various tissues were collected (whole brain, pons/medulla, cerebellum, striatum, hypothalamus/thalamus, olfactory bulb, seminal vesicles, ovaries, eye, kidney, adrenal gland, heart, spleen and lung). Each of the tissue samples (approximately 50-100 mg) were homogenized in 1 ml of TRIZOL reagent (Invitrogen) using a tissue homogenizer. The samples were then incubated at room temperature for 5 min to enhance and ensure the dissociation of nucleoprotein complexes. 200 ul of chloroform was added and the samples were vortexed, incubated at room temperature for 3 min and then centrifuged at 12,000 x g for 15 min at 4°C. The aqueous layer was collected and 500 u.1 of isopropyl alcohol added. The samples were incubated at room temperature for 10 min and centrifuged at 12,000 x g for 10 min at 4°C and the RNA pellet was washed with 1 ml of 75% EtOH made with DEPC-dH20. Samples were mixed by vortexing and centrifuged at 7,000 x g at 4°C for 5 min. The pellet was then allowed to dry for 5-10 min and resuspended in DEPC-dH20. RNA samples were then incubated at 55°C for 10 min to ensure that the RNA was fully in solution. The RNA concentration was determined by spectrophotometery (A26o) and the integrity of the sample was determined by gel electrophoresis. PCR Amplification Procedures RT-PCR Reverse transcription-PCR was performed throughout this study using the following method. First strand cDNA for PCR amplification was prepared by annealing 15 pmol of the downstream 59 gene-specific primer to 1 ug of total RNA. This was accomplished by adding to a PCR tube 1 ul of RNA (1 ug/ul), 1 ul downstream antisense primer (15 pmol/ul) and dH20 up to a final volume of 8.5 ul. The sample was then heated to 80°C for 10 min, put on ice for 5 min and spun in a microfuge to collect the contents of the tube. While still on ice, 11.5 ul of a cocktail consisting of 4 ul 1st strand buffer (5X), 2 ul dNTPs (10 mM each), 2.5 ul DTT (100 mM), 1 ul RNase inhibitor (10 U) (RNasin, Invitrogen) and 2 ul Superscript II reverse transcriptase (400 U) (Invitrogen) was added to the tube. The reaction was then incubated at 42°C for 90 min followed by 95°C for 15 min. Samples were then put on ice for 5 min and spun in a microfuge to collect the contents of the tube. 1 ul of the first strand cDNA was used in PCR amplification using a gene-specific sense and antisense primer set (see below; PCR). In many instances, 10 ul of the RT-PCR product was subjected to Southern blot analysis and membrane hybridization using an internal primer that was radioactively labeled with 3 2P. Positive amplification products were subcloned into the pGEM-T Easy or pBluescript KS vectors and their DNA sequence determined. PCR PCR was performed throughout this study using the following general method. In general, 1 ul of either first strand cDNA or double stranded DNA was used as the template. The actual concentration of template was varied as required. 1 ul of template was added to a PCR reaction cocktail consisting of 5 ul PCR buffer (10X), 1 ul dNTPS (10 mM each), 1 ul each sense and antisense primer (10 pmol/ul), 1 ul HotStarTaq polymerase (5 U) (Qiagen) or 1 ul Pfu Turbo DNA polymerase (2.5 U) (Stratagene) and dH20 up to a final volume of 50 ul. To the top of the PCR reaction was added 30 ul of mineral oil. The mixture was first denatured for 15 min at 95°C if the HotStarTaq polymerase was used or for 3 min at 95°C if the Pfu Turbo DNA 60 polymerase was used and samples were usually amplified for 35 cycles in steps consisting of a 30 s denaturation step at 95°C, a 30 s annealing step at x°C and y seconds extension step at 72°C, followed by 10 min incubation at 72°C, where x = 5°C below the predicted melting temperature of the primer set and y = 60 s/kb of amplification product. 5' RACE The procedure used to perform 5' RACE was as suggested in the 5' RACE System (Invitrogen). All of the reagents used (except KH primers) were included in the kit. Reverse transcription was preformed on total RNA from various tissues from different organisms. Briefly, to a PCR tube was added 2.5 pmol of the downstream most antisense primer, 1 ug of total RNA and dH20 up to a final volume of 15.5 ul. The mixture was incubated at 70°C for 10 min, put on ice for 1 min and spun in a centrifuge to collect the contents of the tube. A reaction mixture consisting of 2.5 ul of PCR buffer (10X), 2.5 ul MgCl 2 (25 mM), 1 ul dNTP (10 mM each), 2.5 ul DTT (100 mM), 0.5 ul of RNase inhibitor (5U) (RNasin) and 0.5 ul of Superscript II reverse transcriptase (100 U) was added and incubated at 42°C for 1 hr. The reaction was then placed at 70°C for 15 min to inactivate the enzymes and terminate the reaction, at which time 1 ul of RNase H (2 U) was added and the mixture was incubated at 37°C for 30 min. The first strand cDNA was then purified through a GlassMax spin cartridge as detailed by the manufacturer. A poly(C) tail was added to the first strand cDNA by combining 6.5 ul dH20, 5 ul of tailing buffer (5X), 2.5 ul dCTP (2 mM) and 10 ul of purified first strand cDNA. The mixture was then heated to 95°C for 3 min, placed on ice for 1 min and the contents of the tube collected by centrifugation. 1 ul of terminal transferase (15 U) was then added and the reaction was incubated at 37°C for 10 min and then at 65°C for 10 min. 5 ul of poly(C) cDNA was amplified with 2 ul of AAP primer (10 uM) and 20 pmol of nested antisense primer for 35 cycles 61 consisting of a 30 s denaturation step at 95°C, a 30 s annealing step at x°C and y seconds extension step at 72°C, followed by 10 min incubation at 72°C. A second round of amplification was performed on 1 ul of the previous PCR reaction (diluted 1:1000) using primers AUAP and the nested antisense primer. 35 cycles consisting of a 30 s denaturation step at 95°C, a 30 s annealing step at x°C and y seconds extension step at 72°C, followed by 10 min incubation at 72°C was performed. For each of the aforementioned PCR amplification reactions, x = 5°C below the predicted melting temperature of the primer set and y = 60 s/kb of amplification product. 10 ul of the second round amplification product was usually subjected to Southern blot analysis and membrane hybridization using an additional nested primer that was radioactively labeled with 3 2P. Positive amplification products were subcloned into the pGEM-T Easy or pBluescript KS vectors and their DNA sequence determined. Site-Directed Mutagenesis The procedure used to perform site-directed mutagenesis was as outlined in the QuikChange Site-Directed Mutagenesis Kit (Stratagene). In general, between 5 and 50 ng of template to be mutagenized was used in each reaction. 1 ul of template was added to a PCR reaction cocktail consisting of 5 ul PCR buffer (10X), 1 ul dNTPS (10 mM each), 1 ul of each primer (10 pmol/ul), 1 ul Pfu Turbo DNA polymerase (2.5 U) (Stratagene) and dH20 up to a final volume of 50 ul. To the top of the PCR reaction was added 30 ul of mineral oil. The mixture was first denatured for 30 s at 95°C and then amplified for 12 cycles consisting of a 30 s denaturation step at 95°C, a 30 s annealing step at 55°C and y seconds extension step at 68°C, where y = 2 min/kb of template DNA. After the 12 rounds of amplification, 2.5 ul of Dpn I (25 U) was added and incubated at 37°C for 1 hr. 1 ul of the sample was then transformed into XL-1 (Blue) cells, plated onto LB agar plates containing the appropriate amount of antibiotic and 62 single recombinant clones containing the desired mutation were identified by restriction enzyme and DNA sequence analyses. Southern Blotting RT-PCR or 5'RACE products were resolved on 2% agarose gels which were subsequently incubated in 0.25 M HCI for 15 min, rinsed in dHzO for 40 min (changing the dHaO approximately every 10 min), soaked in 0.5 M NaOH/1.5 M NaCI for 15min, rinsed in CIH2O for 40 min (changing the CIH2O approximately every 10 min), soaked in 1 M N H 4 O A C for 30 min and blotted to Nytran Supercharge nylon membrane (Schleicher & Schuell). Nytran was prepared by first wetting in dH20 followed by soaking in 1M N H 4 O A C for 15 min. The treated agarose gel was placed between two pieces of prepared Nytran, 3M Whatman paper and paper towels all cut to match the size of the gel. The DNA was transferred overnight at room temperature to the Nytran membrane and the filter was then allowed to dry and subjected to membrane hybridization using specific radioactively labeled 32P-probes. Colony Blots Colony blots were occasionally used for the rapid screening of bacteria transformed with recombinant plasmids that could not be identified through blue-white selection. Using sterile toothpicks, bacterial colonies were transferred to Hybond-N nylon membranes (Amersham) that were placed on LB agar plates containing the appropriate antibiotic. Each bacterial colony was numbered and duplicated onto another stock LB agar plate. After 24 hr of growth at 37°C, the membranes were removed, incubated for 2 min in 0.5 M NaOH, then in 1 M Tris pH 7.5 for 5 min and finally in 1.5 M NaCl/0.5 M Tris pH 7.5 for 5 min. Membranes were then allowed to fully dry and subjected to membrane hybridization using specific radioactively labeled 3 2P-probes. 63 Generation of Radioactive Probes and Membrane Hybridization For randomly primed probes, approximately 200 ng of gel purified DNA was typically used. The DNA was brought up in dEbO to a final volume of 20ul, incubated at 90°C for 5 min, placed on ice and the contents of the tube collected by centrifugation. The labeling cocktail contained 15 ul Random primer buffer (Invitrogen), 2 ul dTTP, 2 ul dGTP, 5 ul [a-32P] dATP, 5 ul [a-32P] dCTP (10 mCi/ml, Amersham), 1 ul Klenow DNA polymerase (3-9 U) (Invitrogen) and ( IH2O to a final volume of 50 ul was added. The sample was incubated at room temperature for 90 min and then unincorporated a-32P was removed by passing the sample through a G50 (Pharmacia) spin column. The probe was then heated to 90°C for 10 min, placed on ice for 5 min and the contents of the tube were collected by centrifugation. The probe was then added to the hybridization buffer. For end-labeled oligonucleotide probes, the sample cocktail consisting of 1 ul oligonucleotide (7 pmol), 4 ul kinase buffer (5X, Invitrogen), 2 ul [y-32P] dATP (10 mCi/ml, Amersham), 1 ul T 4 polynucleotide kinase (10 U) (Invitrogen) and dH^O to a final volume of 20 ul and was incubated at 37°C for up to 4 hr. Unincorporated y-32P was removed by passing the sample through a G25 spin column (Pharmacia). The probe was then added to the. hybridization buffer. Membranes were prehybridized in hybridization buffer (5X Denhardt's solution (50X; 5 g of Ficoll (Type 400, Sigma) 5 g of polyvinylpyrrolidone, 5 g of bovine serum albumin (Fraction V, Sigma) in 500 ml dH20)), 5X SSPE (20X; 175.3 g/1 NaCI, 27.6 g/1 NaH2P04H20, 7.4 g/1 EDTA, pH 6.8), 0.3% SDS for at least 30 min at the desired hybridization temperature (65°C for randomly primed probes and 37°C for end-labeled oligonucleotide probes). After prehybridization, the buffer was replaced by fresh hybridization buffer and the radioactive probe was added. Hybridization was performed overnight at the appropriate temperature in a water bath 64 with gentle agitation. After hybridization, the membranes were washed with several changes of wash buffer (2X SSPE, 0.3% SDS) at the hybridization temperature until there was no detectable background. If necessary, in order to reduce the background, the SSPE concentration in the wash buffer was reduced to IX or 0.5X. Membranes were then air dried and exposed to film (Biomax MS double emulsion film, Kodak) at -80°C in the presence of an intensifying screen for varying times depending on the signal intensity. Screening of a A.ZAPII Rat Brain cDNA Library Rat-nca sequences were isolated from a A Z A P I I rat brain cDNA library enriched for cDNAs greater than 4 kb (Snutch et al., 1990). LB growth media contained (in g/1 of media): tryptone (10), yeast extract (5) and NaCI (10). X dilution buffer (X dil) contained in (ml/100 ml) 1 M Tris pH 7.5 (1) and 1 M MgS0 4 (2). Culture plates consisted of NZYCM growth media containing (in g/1) NZ amine (10), NaCI (5), Casamino acids (1), yeast extract (5), MgS0 4 (2) and agar (14).Top agarose consisted of NZYCM media containing 2% agarose. Four rounds of plaque purification were performed. The first round was carried out on 150 x 15 mm culture plates and all subsequent rounds were carried out on 100 x 15 mm plates. The probe was generated by amplifying a 646 bp RT-PCR product from rat whole brain total RNA with the primer set KH15-KH16. The RT-PCR product was run out on a 2% agarose gel, subjected to Southern blotting and probed with the internal primer KH14 that had been y-32P labeled. The RT-PCR product was then subcloned into pGEM-T Easy and the DNA sequence was determined. The 646 bp fragment was excised with Eco RI, gel purified and served as a radiolabeled DNA probe. Ten 150 x 15 mm NZYCM plates containing approximately 750,000 pfu were screened in the first round of plaque purification. Plaques were transferred to Hybond-N nylon membranes (Amersham) by capillary action. Duplicate membranes were prepared for each plate. The 65 membranes were prepared for hybridization by soaking in 0.5 M NaOH/1.5 M NaCI for 2 min, then in 1 M Tris pH 7.5 for 5 min, then in 2X SSPE for 5 min and then allowed to air dry. The membranes were then prehybridized and hybridized in 5X SSPE, 5X Denhardt's solution, 0.3% SDS and incubated overnight at 65°C with the radiolabeled DNA probe. Membranes were then washed in 2X SSPE, 0.3% SDS at 65°C, air dried, and exposed to film (Biomax MS double emulsion film, Kodak) for 24 hr at -80°C with an intensifying screen. Plaques that were positive on both the original and duplicate membranes were extracted from the agar with a pipette tip and placed in 0.5 ml of X dil. 0.5 ml of chloroform was added to the tube and mixed well. The phage from these positive plugs were diluted in X dil for the second round of purification. This procedure was repeated for an additional three more times and samples were considered pure when 100% of the plaques on a plate gave a hybridization signal. Stocks of pure phage were then established and stored under chloroform at 4°C. To excise the cDNAs for the A Z A P H vector, 200 ul of XL-1 (Blue) bacteria (OD 6 0 0 ~ 1.0), 50 ul of phage stock (containing > 1 X 105 phage particles) and 1 ul of ExAssist helper phage (> 1 X 106 pfu/ml, Stratagene) were added together and incubated at 37°C for 15 min. Three ml of 2X YT media was then added and placed in a shaker incubator for 2 hr at 37°C. The mixture was then heated to 70°C for 20 min and then centrifuged for 15 min. The supernatant was then transferred to a new tube and contained the cDNA of interest packaged as a filamentous phage particle and was stored at 4°C. The rescued phagemid was plated by combining 1 ul of this stock and 200 ul of SOLR host cells (ODeoo ~ 1.0). This mixture was incubated at 37°C for 15 min and then 100 ul of the mixture was plated onto LB agar plates containing 100 ug/ml ampicillin. Single colonies were picked and analyzed and the cDNAs of interest were subsequently reintroduced into XL-1 (Blue) cells. 66 Constructs Generated for This Study cDNA Clones nca-1 The C . elegans clone "nca-1 5' RACE" was obtained by 5' RACE using N2 total RNA. Primer KH80OS was used to reverse transcribe the RNA. The nested primer KH80 was used in the subsequent PCR steps and the furthest upstream primer KH104 was used as a probe for Southern blotting. A single positive PCR product was subcloned into the pBluescript KS vector cut with Eco RV. The start site of nca-1 was deduced from this clone by DNA sequencing. The clone "B" was obtained by RT-PCR using N2 total RNA and the primer KH108 for first strand cDNA synthesis and the KH106-KH117 primer set for PCR amplification. The 877 bp product, spanning from bp 1-877, was subcloned into the pGEM-T Easy vector and sequenced. The clone "N1-A3" was generated by PCR using the primer set KH107-KH108 and a C . elegans yeast-two-hybrid library (provided by R. Barstead, C . elegans Gene Knockout Project) as a template. The 1346 bp PCR product (spanning from bp 2012-3357) was subcloned into the pGEM-T Easy vector and its DNA sequence determined. Clone "N1-A3" had a single PCR-induced error that was repaired using the primer set KH119-KH120 and the QuikChange Site-Directed Mutagenesis kit (Stratagene) and was renamed "N1-A3SDM'. The clone "N1-A4" was generated by RT-PCR using N2 total RNA and the primer KH87 for first strand cDNA synthesis and the KH107-KH108 primer set for PCR amplification. The resulting PCR product was subcloned into the pBluescript KS vector cut with Eco RV and its DNA sequence determined. The cDNA insert was found to contain a 102 bp insertion at bp 2224 and a 5 bp deletion further downstream. The deletion was repaired by swapping the erroneous 67 sequence from "N1-A4" with the correct sequence from "N1-A3SDM" and was renamed "fixed N1-A4". The yk207g5 and yk354d9 cDNA clones were obtained as A.ZapH clones from Y. Kohara (C. elegans cDNA Project). The pBluescript SK plasmids containing the cDNA inserts were excised from A Z A P I I following the protocol outlined above and their DNA sequence determined. The 2300 bp insert in the yk207g5 cDNA clone spanned from bp 267-2566. The 3214 bp insert in the yk354d9 cDNA clone spanned from bp 2446 to the poly(A) tail at bp 5558 and also contained an apparent 101 bp intron upstream of the Hind III site at bp 3302. The Full-Length "nca-1" cDNA Two different full-length nca-1 cDNAs were generated by piecing together the overlapping cDNA clones shown in Figure 7. To generate the smaller full-length cDNA clone, "nca-r, the overlapping cDNA clones "B", yk207g5, "N1-A3SDM" and yk354d9 were pieced together in a three step process. The first step involved joining yk207g5 and yk354d9 taking advantage of the two Hind HI sites (at bp 2042 and bp 3302, respectively) in the "nca-1" cDNA. The two cDNAs were joined together using Eco RI in the upstream MCS in pBluescript SK vector and Hind HI and also removed the intron in yk354d9 during the process. This new cDNA was named "2-3" and spanned from bp 267-5558, but lacked the sequence between the two Hind UI sites. The second step involved adding cDNA clone "B" onto cDNA clone "2-3" by using a Not I sequence in the upstream MCS of both the pGEM-T Easy and pBluescript SK vectors and the Swa I site at bp 814. This new cDNA clone was named "B-2-3" and spanned from bp 1-5558, but was missing the Hind IE-Hind III fragment. The third step involved inserting the sequence between the two Hind III sites into the cDNA clone "B-2-3" using the cDNA clone "N1-A3SDM". Orientation of the inserted Hind III fragment was determined by restriction 68 Figure 7. Diagram Illustrating the Cloning Strategy Used to Generate Full-Length nca-1 cDNAs The corresponding region of the NCA-1 protein is shown above the cDNA clones. Transmembrane segments are indicated as boxes and the cytoplasmic and extracellular domains are indicated as lines. The full length "nca-1" cDNA was generated by piecing together the partial cDNA clones "B", yk207g5, "N1-A3SDM" and yk354d9 using the restriction enzymes Swa I and Hind III. The full length "nca-1 AS" cDNA was generated by piecing together the partial cDNA clones "B-2-3" and "fixed N1-A4" using the restriction enzyme Hind III. The additional 102 bp of coding sequence in the "fixed N1-A4" clone and the extra 34 aa in the corresponding "NCA-1 AS" protein are represented as triangles on the diagram. 69 ss ss © 0 <• <• I I • i ca x I CZZ1 c '3 S o Q g S o Q 2 N H N H •St. 8 S a •a 85 r ~ ~ i r~~i i . i i * i i * i r ~ i c 6 o Q c "55 •». .1 Ml •a s — s I I 03 T o Q O H • < O H • 8 < 70 enzyme analysis. The newly assembled full-length cDNA clone in pBluescript SK (bp 1 -5558) was called, "nca-1", and was sequenced in its entirety. The full-length "nca-1" cDNA was excised from pBluescript SK with Not I (upstream MCS) and Apa I (downstream MCS) and introduced into these sites in pCDNA3.1 zeo. In the "nca-V cDNA clone the start site is at bp 12 and the stop site is at bp 5403. The Full-Length "nca-1 AS" cDNA To generate the larger full-length nca-1 cDNA, "nca-1 AS", the Hind HI fragment from the cDNA clone "fixed N1-A4" was introduced into the cDNA clone "B-2-3" and the orientation of the inserted Hind III fragment was determined by restriction enzyme analysis. The newly assembled full-length cDNA clone in pBluescript SK was called "nca-1 AS" and was sequenced in its entirety. The full-length "nca-1 AS" cDNA was then excised from pBluescript SK with Not I (upstream MCS) and Apa I (downstream MCS) and introduced into those sites in pCDNA3.1 zeo. The "nca-1 AS" cDNA clone only differed from the "nca-1" cDNA by the addition of the 102 bp alternatively spliced sequence. nca-2 The clones "Ul" and "fixed LI" were generated by RT-PCR using N2 total RNA and an oligo dT primer for first strand cDNA synthesis and the SL1-KH82AOS primer set for PCR amplification. Two different PCR products were amplified, an 888 bp product called "Ul" and a 770 bp called "LI". Both PCR products were subcloned into the pGEM-T Easy vector and the DNA sequence determined. "Ul" was found to span from bp 1-853 (an additional 35 bp of SL1 oligo sequence was present at the 5' end of the PCR product) and "LI" spanned from bp 119-853 (an additional 35 bp of SL1 oligo sequence was present at the 5' end of the PCR product). "LI" also appeared to contain PCR-induced errors that were subsequently corrected by swapping the 71 erroneous sequence from "LI" with the correct sequence from "Ul" and was renamed "fixed LI" The "115-116 PCR" clone was generated by PCR using the primer set KH115-KH116 and a C. elegans yeast-two-hybrid library (provided by R. Barstead, C. elegans Gene Knockout Project) as a template. The 1923 bp PCR product (spanning from bp 309-2231) was subcloned into the pGEM-T Easy vector and the DNA sequence determined. The yk24d9 cDNA was obtained as a AZAPII clone from Y. Kohara (C. elegans cDNA Project). The pBluescript SK plasmid containing the cDNA insert was excised from A.ZAPII following the protocol outlined above and its DNA sequence determined. The 3797 bp insert in the yk24d9 cDNA clone spanned from bp 1834 to the end of the poly(A) tail at bp 5630. The Full-Length "nca-2" cDNA Two different full-length nca-2 cDNAs were generated by piecing together the overlapping cDNA clones shown in Figure 8. To generate the smaller of the nca-2 cDNAs, the cDNA clones "fixed LI", "115-116 PCR" and yk24d9 were pieced together in a two step process. The first step involved joining "115-116 PCR" to "fixed LI" using the common Eco RV site (at bp 350) and a downstream Pst I site in the MCS of the pGEM-T Easy vector. This new cDNA clone, named "5' nca-2", spans from bp 119-2231. The second step involved joining "5' nca-2" to yk24d9 using the common Cla I site (at bp 2118) and Sst II sites present upstream in the MCS of both the pGEM-T Easy and pBluescript SK vectors. The newly assembled full length clone in pBluescript SK (bp 119-5630) was named, "nca-2", and was sequenced in its entirety. In the "nca-2" cDNA clone the start site is at bp 157 and therefore is downstream of the Sal I site (at bp 147). The stop codon is at bp 5446. The full length ^nca-2" cDNA was then excised from pBluescript SK with Sal I and Xho I (downstream MCS) and introduced into the Sal I site in pCDNA3.1 zeo. Orientation was determined by restriction enzyme analysis. 72 Figure 8. Diagram Illustrating the Cloning Strategy Used to Generate Full-Length nca-2 cDNAs The corresponding region of the NCA-2 protein is shown above the cDNA clones. Transmembrane segments are indicated as boxes and the cytoplasmic and extracellular domains are indicated as lines. The full length "nca-2" cDNA was generated by piecing together the partial cDNA clones "fixed LI", "115-116 PCR" and yk25d9 using the restriction enzymes Eco RV and Cla I. The full length "A5' nca-2" cDNA was generated by piecing together the partial cDNA clone "Ul" and "nca-2" using the restriction enzyme Sal I. The additional 66 bp of coding sequence in the "Ul" clone and the extra 22 aa in the corresponding "A5' NCA-2" protein are indicated by green boxes. 73 7 4 The Full-Length"A5' nca-2" cDNA To generate the larger full-length nca-2 cDNA, "A5' nca-2", the 5' end of cDNA clone "nca-2" was replaced with a region from "Ul" using an upstream Sst JJ site present in the MCS of both the pGEM-T Easy and pBluescript SK vectors and the Sal I site at bp 147, generating the full length cDNA clone "A5' nca-2" (bp 1-5630). The resulting cDNA was sequenced in its entirety. In this clone the start site is upstream of the Sal I at bp 91. The "A5' nca-2" cDNA was excised from pBluescript SK with Sst II (upstream MCS) and Xho I (downstream MCS) and introduced into these sites in pSL 1180. The cDNA was then excised again out of pSL 1180 with Pst I (upstream MCS) and Xho I and introduced into the same sites in pCDNA3.1 zeo. rat-nca The clone "5' RACE xat-nca" was obtained by 5' RACE using whole rat brain total RNA. Primer KH47A was used to reverse transcribe the RNA. The nested primer KH85 was used in the subsequent PCR steps. The furthest upstream primer, KH73, was used as a probe for Southern blotting. A single positive PCR product was subcloned into the pGEM-T Easy vector and sequenced and the start site of xat-nca was deduced from this clone. The clone "KH15-KH16" was obtained by RT-PCR using whole rat brain total RNA and the primer KH15 for first strand cDNA synthesis and the KH15-KH16 primer set for PCR amplification. The 646 bp product, spanning from bp 2883-3528, was run out on a 2% agarose gel, subjected to Southern blotting and probed with the internal primer KH14 that had been y-32P labeled. The RT-PCR product was then subcloned into pGEM-T Easy and the DNA sequence determined. The xat-nca clones "Ul" and "U2" were obtained by RT-PCR using rat brain total RNA and the primer KH83 for first strand cDNA synthesis and the upstream KH61-KH84 primer set for PCR amplification. The RT-PCR products were run out on a 2% agarose gel, subjected to 75 Southern blotting and probed with the internal primer KH62 that had been y- P labeled. The RT-PCR products were then subcloned into pGEM-T Easy and their sequences were determined. The smaller product, named "U2" was a 424 bp PCR product that spanned from bp 4785-5208. The larger product, named "Ul" was a 520 bp PCR product that was the same as "U2" except for a 96 bp insertion at bp 5101. The cDNA clones, "10-2111", "9-1111" and "1-2111" were obtained by screening a A Z A P H rat brain cDNA library with the insert of clone "KH15-KH16" utilized as a 3 2P-radiolabeled probe. The pBluescript SK plasmid containing the cDNA inserts were excised following the protocol outlined above and sequenced. The 5361 bp insert in the "10-2111" cDNA clone spanned from bp 1-5361, with the start site at bp 78 and stop codon at bp 5292. The 5066 bp insert in clone "9-1111" spanned from bp 493-5558 and the 3639 bp insert in clone "1-2111" spanned from bp 3333-6791. The Full-Length "rat-nca A, F10-9" cDNA Since clone "10-2111" appeared to be full-length and to span both the start and stop codons, it was used as the template for generating the full-length "rat-nca A" cDNA. Clone "10-2111" was then excised from pBluescript SK at the Xba I sites present in the upstream MCS of pBluescript SK and at bp 5290 and introduced into the Xba I site in pCDNA3. Orientation was determined by restriction enzyme analysis. Clone "10-2111" in pCDNA3 was sequenced in its entirety and was found to have two in-frame 75 bp deletions spanning from bp 1842-1916 and from bp 3768-3842, respectively. Consequently, these two deletions were repaired and the full length "rat-nca A" cDNA, "F 10-9", was generated from the following multi-step process (Figure 9). The first step involved building the cDNA construct, "5' PCR 10-2111" that had a shorter 5' UTR than clone "10-2111". This was accomplished by PCR using the primer set 76 Figure 9 . Diagram Illustrating the Cloning Strategy Used to Generate the Full-Length rat-itca A cDNA The corresponding region of the "rat-NCA A" protein is shown above the cDNA clones. Transmembrane segments are indicated as boxes and the cytoplasmic and extracellular domains are indicated as lines. The full length "rat-nca A" cDNA was generated by piecing together the partial cDNA clones "5' PCR 10-2111", "10-2111" and "9-1111" using the restriction enzymes Pst I, Nde I, Eco RI, Afl II, and Xba I. The 75 bp deletion between bp 3768-3842 present in clone "FL 10-2111" is represented as a red box over the corresponding sequence. 77 78 KH73-KH50A and the clone "10-2111" in pBluescript SK as a template. The KH-73 sense primer was designed to include a Not I site to facilitate future cloning. The 920 bp PCR product (spanning from bp 67-976) was subcloned into the pGEM-T Easy vector and the DNA sequence determined. The second step was to extend the clone "5' PCR 10-2111" in the 3' direction by adding a Pst l-Nde I fragment from clone "10-2111" in pBluescript SK. This was accomplished by using the common Pst I site at bp 329, the Nde I site at bp 1783 and the Nde I site in the downstream MCS of pGEM-T Easy. The resulting cDNA clone was called "10-2111 LPCR" and spanned from bp 67-1783. The third step involved adding the correct sequence between the two Nde I sites (at bp 1783 and bp 4494, respectively) to the clone "10-2111 LPCR" using the clone "9-1 111". Orientation of the insert was determined by restriction enzyme analysis and the resulting cDNA was named "10-2111 VLPCR" and spanned from bp 67-4494. The fourth step involved using "10-2111 VLPCR" to repair the 75 bp deletion spanning from bp 1842-1916 present in the clone "10-2111" in pCDNA3. This was accomplished by replacing the sequence between two Eco RI sites present in the clone "10-2111" in pCDNA3 with the corresponding sequence from clone "10-2111 VLPCR" using the Eco RI site present in the upstream MCS of pCDNA3 and pGEM-T Easy, respectively and the common Eco RI site at bp 2113. This repaired the 75 bp deletion spanning from bp 1842-1916, but not the 75 bp deletion from bp 3768-3842. This partially repaired cDNA was called "FL 10-2111" and spanned from bp 67-5295. The last step involved replacing the 75 bp deletion from bp 3768-3842 present in "FL 10-2111". This was accomplished by exchanging the erroneous sequence in "FL 10-2111" with the correct sequence from clone "9-1 111" using the Afl II site at bp 2264 and the Xba I site at bp 5290. The upstream Xba I site originally present in clone "10-2111" in pCDNA3 was lost in the 79 making of clone "FL 10-2111" in pCDNA3. The resulting clone, in pCDNA3, was now the full-length "rat-nca A" cDNA and was named, "F 10-9". The "rat-nca A" cDNA spans from bp 67-5295 with start and stop codons at bp 78 and 5292, respectively. The Full-Length "rat-nca B, FL Ul" cDNA A multi-step process was used to assemble the full-length "rat-nca B" cDNA clone (Figure 10). The first goal was to extend the clone "Ul" from bp 5208 to bp 5361 using clone "10-2111" in pBluescript SK. Clone "Ul" has a 520 bp insert that spans from bp 4785-5208, as well as a 96 bp insertion at bp 5101 that contains an additional Hpa I. Thus, clone "Ul" contains two Hpa I sites, one at bp 5135 and one at bp 29 in the 96 bp insertion. In order to facilitate future cloning steps, clone "Ul" was cut and recircularized with Hpa I and then Sal I. The resulting clone, "UHS", spanned from the Sal I site at bp 5035 to bp 5208, but was missing the sequence between the two Hpa I sites. The second step was to extend the clone "UHS" to bp 5361 by adding on a Hpa l-Eco RI piece from clone "10-2111" in pBluescript SK. This was accomplished by using the common Hpa I site at bp 5135 and the Eco RI sites in the downstream MCS of pGEM-T Easy and pBluescript SK, respectively. The resulting cDNA clone was called "UHS HE" and spanned from bp 5035-5361, but was missing the Hpa l-Hpa I sequence. The third step involved introducing the missing Hpa l-Hpa I sequence in clone "UHS HE" using clone "Ul". Orientation of the insert was determined by restriction enzyme analysis and the clone was sequenced in its entirety. The newly assembled partial cDNA was called "Ul-STOP" and spanned from bp 5035-5361 and contained the 96 bp insert at bp 5101. The fourth step involved assembling the full-length "rat-nca B" clone in pBluescript KS. This was accomplished by excising a Not l-Sal I (Not I site in primer KH73 and Sal I at bp 5035) 80 Figure 10. Diagram Illustrating the Cloning Strategy Used to Generate the Full-Length rat-wca B cDNA The corresponding region of the "rat-NCA B" protein is shown above the cDNA clones. Transmembrane segments are indicated as boxes and the cytoplasmic and extracellular domains are indicated as lines. The full length "rat-nca B" cDNA was generated by piecing together the partial cDNA clones "Ul", "10-2111" and "F 10-9" using the restriction enzymes Sal I and Hpa I. The additional 96 bp of coding sequence in the "Ul" clone and the extra 32 aa in the corresponding "rat-NCA B" protein are represented as triangles on the diagram. The inset is an enlargement of clone "Ul" and outlines the cloning strategy used to extend this clone in the 3' direction. 81 82 fragment from clone "F 10-9" and subcloning into these same sites in pBluescript KS. This intermediate clone spanned from bp 67-5035. The next step involved adding the sequence from clone "Ul-STOP" to this intermediate clone using the shared Sal I site at bp 5035 and the Apa I sites in the downstream MCS of pGEM-T Easy and pBluescript KS, respectively. The newly assembled full-length "rat-nca B" cDNA clone in pBluescript KS (bp 67-5361) was called, "FL Ul" and was sequenced in its entirety. The full-length "rat-nca B" cDNA was then excised from pBluescript KS with Not I (in primer KH73) and Apa I (downstream MCS) and introduced into these same sites in pCDNA3.1 zeo. In the "rat-nca B" cDNA clone, the start and stop codons are at bp 78 and 5292, respectively and there is a 96 bp of extra coding sequence (from an alternatively spliced exon) inserted at bp 5101 . rat-ncaA::FLAG, "F10-9::FLAG" The FLAG epitope (DYKDDDDK) was introduced into the "rat-nca A" cDNA as a carboxyl terminal fusion. The strategy of introducing the FLAG epitope took advantage of the in-frame blunt cutter Eco RV at bp 5288, that would cut the clone exactly one codon upstream of the stop site. Furthermore, the Eco RV site would be destroyed by the introduction of the FLAG sequence and would facilitate the isolation of recombinant clones. Finally, the introduction of the FLAG sequence would also introduce an additional Bgl II site. To generate a double-stranded FLAG insert, 50 pmol of the primers Flag S and Flag A were mixed (in 50 mM Tris pH 8.0,10 mM MgCl 2, 50 mM NaCI) in a total volume of 10 ul, heated to 65°C for 10 min, placed on ice for 5 min and then centrifuged to collect the contents of the tube. The "rat-nca A" clone, "F 10-9" in pCDNA3 was then digested with Eco RV for 2 hrs at 37°C, heated to 65°C for 30 min, placed on ice for 5 min and then briefly centrifuged. The ligation of the FLAG epitope to the Eco RV digested "rat-nca A" clone was accomplished by adding 50 ng of Eco RV digested "rat-nca A", 2 ul 5X ligase buffer (Invitrogen), 4 ul ds FLAG 83 mixture, 1 ul T4 DNA ligase (1 U) (Invitrogen) and dH20 up to 10 ul. The ligation reaction was then incubated at 20°C overnight. The ligation reaction was then ethanol precipitated, resuspended in dH20 and digested with Eco RV for 1 hr at 37°C. 1 ul of this digestion was transformed into XI-1 (Blue) cells and plated onto LB agar plates containing 100 ug/ml ampicillin. Putative positive clones were determined by restriction enzyme analysis. Positive clones that lacked an Eco RV site and contained an additional Bgl II site were examined further. The orientation of the FLAG insert was determined by PCR using the primer set KH62-Flag A. Positive clones were then sequenced with primer KH62 to ensure that the FLAG epitope was in-frame with respect to "rat-nca A". The resulting "rat-nca A::FLAG" fusion was called, "F 10-9::FLAG". Promoter::GFP Fusion Constructs p«ca-i::GFP In order to examine the cellular expression pattern of nca-7, the nca-1 promoter::GFP fusion construct was generated (Figure 11 A). The clone "CI 1D2.6 PCR" was generated by two rounds of PCR. The first round of PCR used the primer set KH79OS-KH80OS and N2 genomic DNA as a template. The second round of PCR used the primer set KH79-KH80 and 1 ul of the first round PCR reaction as a template. The KH79 primer was designed so that it contained a Sph I site and that it annealed 3418 bp upstream of the start site of nca-7. The KH80 primer was designed so that it contained aXba I site (that would make the insert in-frame with respect to GFP) and so that it annealed to exon 1 of nca-7 downstream of the start site. The 3516 bp PCR product was subcloned into pGEM-T Easy and its DNA sequence was determined. The insert was then excised from pGEM-T Easy using the Sph I sites in the insert and in the MCS on the 84 Figure 11. Diagrams Illustrating the Genomic Region Used to Construct the p«ca-2::GFP and p«ca-2::GFP Reporter Constructs For all gene diagrams, exons and introns are represented as boxes and lines, respectively. A) The pnca-l::GFP reporter construct contains 3418 bp of putative promoter region upstream of the start site in exon 1 and has GFP fused in-frame with respect to the nca-1 open reading frame at exon 1. B) The p«ca-2::GFP reporter construct contains 3345 bp of putative promoter region upstream of the start site in exon la and has GFP fused in-frame with respect to the nca-2 open reading frame at exon 3. 85 86 opposite side of pGEM-T Easy and introduced into the Sph I site in pPD95.67. Orientation of the insert was determined by restriction enzyme analysis and the insert was brought into frame with respect to GFP by digesting and recircularizing with Xba I. The reading frame with respect to GFP was further verified by sequencing and the clone was named pnca-J::GFP. p«ca-2::GFP In order to examine the cellular expression pattern of nca-2, the nca-2 promoter: :GFP fusion construct was assembled in a multi-step process (Figure 1 IB). Clone "1" was generated by two rounds of PCR. The first round of PCR used the primer set KH91-KH94 and N2 genomic DNA as a template. The second round of PCR used the primer set KH92-KH99 and 1 u.1 of the first round PCR reaction as a template. The KH92 primer was designed so that it annealed just upstream of a unique Pst I site. The 1998 bp PCR product was subcloned into pGEM-T Easy and its DNA sequence determined. Similarly, clones "2" and "3" were generated by PCR using the primer sets KH98-KH101 and KH93-KH100, respectively and 1 ul of the KH91-KH94 reaction as a template. The 2845 bp "2" and 1705 bp "3" PCR products were subcloned into pGEM-T Easy and their DNA sequences determined. Clones "1" and "2" overlapped each other by 239 bp and the overlap contained a common unique Eco RI site. Clones "2" and "3" overlapped each other by 170 bp and the overlap contained a common unique Sph I site. The clone "C27F2.3 PCR" was generated by two rounds of PCR. The first round of PCR used the primer set KH810S-KH82AOS and N2 genomic DNA as a template. The second round of PCR used the primer set KH81-KH82A and 1 ul of the first round PCR reaction as a template. The KH82A primer was designed so that it contained a Pst I site (that would make the insert in-frame with respect to GFP) and so that it annealed to exon 3 of nca-2 downstream of the start site. The 3515 bp PCR product was subcloned into pGEM-T Easy and its DNA sequence 87 determined. Clones "3" and "C27F2.3 PCR" overlapped each other by 194 bp and the overlap contained a common unique Xba I site. A multi-step process was used to assemble the full length pnca-2::GFP fusion construct using the unique enzyme sites Eco RI, Sph I md Xba I and the final product was assembled in pBluescript KS. The new clone, called "1-2-3-4 hyb", contained a 9423 bp insert and had 3345 bp of putative promoter region upstream of the start site in exon la. The insert was then excised from pBluescript KS using the Pst I site in the insert just downstream from the KH92 annealing site and in the Pst I site introduced in KH82A and cloned into the Pst I site in pPD95.67. Orientation of the insert was determined by restriction enzyme analysis and the reading frame with respect to GFP was verified by sequencing and the clone was named p«ca-2::GFP. nca-1 Promoter: :DsRed2 Fusion Construct In order to examine whether nca-1 and nca-2 are coexpressed in any cells, the following nca-1 promoter: :DsRed2 fusion construct was assembled in a multi-step process. The first step involved replacing the GFP open reading present in the pPD95.67 vector with that of DsRed2 from the pDsRed2 vector (Clontech). The introduction of the DsRed2 open reading into the pPD95.67 vector took advantage of the fact that both the GFP and DsRed2 open reading frames contained a Kpn I site upstream of their respective start sites and an EcoR I site downstream of their respectivejStop sites. By using these two enzymes not only would the GFP sequence be replaced with that for DsRed2, but the pre-established reading frame for the pPD95.67 vector also would not be altered by the insertion of the DsRed2 coding sequence. The resulting vector was named "pPD95.67 DsRed2", but it lacked the nuclear localization signal (NLS) present in the original pPD95.67 vector. This loss of the NLS in clone "pPD95.67 DsRed2" upon digestion with Kpn I was due to the fact that the NLS present in the pPD95.67 vector is encompassed by two Kpn I sites (at bp 122 and bp 167, respectively). 88 The second step involved inserting the putative promoter region of nca-1 obtained from the clone pnca-1 v.GFP into the "pPD95.67 DsRed2" vector. This was accomplished by using the shared Sph I site present in the MCS of both of these clones and the Kpn I sites at bp 122 and at bp 167 from the clones pnca-l::GFP and "pPD95.67 DsRed2", respectively. The resulting clone was named "pnca-1 pPD95.67 DsRed2" and contained the same putative promoter region that is present in the pnca-1 ::GFP clone but lacked the NLS. The last step was to re-introduce the NLS back into clone "pnca-1 pPD95.67 DsRed2" using the enzyme Kpn I. Orientation of the newly introduced NLS was determined by restriction enzyme analysis, as well as by DNA sequencing. In addition, the reading frame of the nca-1 gene with respect to DsRed2 was also verified by DNA sequencing. The newly generated clone was named p«ca-/::DsRed2 and was identical to that of pnca-l::GFP (Figure 11A) except for the presence of DsRed2 in place of GFP. Identity and Similarity Analysis of Four Domain-Type VGIC am Subunits In order to generate identity trees for the Ca 2 + and Na + channel a<i) subunits, respectively, the predicted amino acid sequence of the four transmembrane domains of the representative classes of either Ca 2 + or Na + channels were aligned pairwise using the default settings of the CLUSTAL W algorithm (Vector NTI Suite 7.0, Invitrogen). From these analyses, sequence identities between each class of Ca 2 + or Na + channel relative to one another were obtained and served as the basis for generating the identity trees. The same approach was used to generate the identity tree for the four domain-type VGIC a(i) subunits, where the predicted amino acid sequences of only the four domains of the NCA channels were compared pairwise to the homologous regions of the different classes of Ca 2 + and Na + channels. Similarly, the same approach was used to obtain the percent similarity and identity between different regions of the 89 NCA channel proteins with respect to one another, as well as the percent similarity and identity of the whole NCA channels relative to each other. In vitro Translation The TNT Coupled Reticulocyte Lysate System (Promega) was used to determine whether the xat-nca A cDNA could produce a full length Rat-NCA A protein product in vitro. Briefly, the reaction contained 25 ul TNT Rabbit Reticulocyte Lysate, 2 ul TNT reaction buffer (25X), 1 ul TNT RNA T7 polymerase (10-20 U), 1 ul amino acid mixture (all amino acids, minus methionine, 1 mM), 2 ul [35S]-methionine (lOmCi/ml, Amersham), 1 ul RNase inhibitor (10 U) (RNasin, Invitrogen), 1 ul rat-nca A cDNA (1 ug/ul) and DEPC-dH20 up to 50 ul. The mixture was incubated at 30°C for 90 min. Unincorporated 35S-methionine was removed by passing the sample through a G25 spin column (Pharmacia). 5 ul of the sample reaction was mixed with 5 ul of 2X SDS-PAGE sample buffer and loaded on a 6% SDS-PAGE gel using established protocols (see below; Western Blotting). The gel was then transferred to 3M Whatman paper, dried using a gel drier (BioRad) and exposed to film (Biomax MR single emulsion film, Kodak) overnight at room temperature. The approximate size of the in vitro translated protein was determined by comparing to pre-stained molecular weight markers (Invitrogen). Protein Isolation from Rat Brain and Human Embryonic Kidney tsA201 Cells In order to obtain rat whole brain protein lysates, an adult female rat was euthanized by cervical dislocation, decapitated and the brain removed and frozen in liquid nitrogen. The frozen brain was ground and homogenized in RIPA buffer (150 mM NaCI, 1% NP-40,0.5% deoxycholate, 0.1% SDS, 50 mM Tris pH 7.5 and a mixture of protease inhibitors (Roche)). The solution was incubated on ice for 1 hr, centrifuged at 15,000 x g for 15 min at 4°C and the 90 supernatant was collected. The total protein concentration was determined using the BCA Protein Assay Kit (Pierce) and 10 ug of protein was loaded onto a 5% SDS-PAGE gel. Proteins were then subjected to Western blot analysis (see below; Western Blotting). To isolate proteins expressed in cultured cells, human embryonic kidney (HEK) tsA201 cells were transiently transfected using Lipofectamine (Invitrogen) and the cDNAs of interest (1 ug each plasmid). Two days post-transfection, cells were lysed in RIPA buffer, incubated on ice for 1 hr and then centrifuged at 15,000 x g for 15 min at 4°C. Both the supernatant and pellet were kept for further analysis. The total protein concentration of both fractions was determined using the BCA Protein Assay Kit (Pierce) and 10 ug of protein was loaded onto a 5% SDS-PAGE gel. Proteins were then subjected to Western blot analysis (see below; Western Blotting). Generation of Antibodies and Immunohistochemistry Fusion Protein Construction To construct the rat-nca::RsaA fusion protein, "KH89 to KH90 pCX-TOPO", the primers KH89 and KH90 were designed such that the rat-nca sequence would be in-frame with the CX leader and the C-terminal, truncated RsaA ORF present in the pCX-TOPO vector (Invitrogen). PCR employing HotStarTaq polymerase (Qiagen) was used to amplify a 165 bp fragment (bp 1059-1223) encoding amino acids 328-382 of the cytoplasmic linker between Domains I and II of rat-NCA. The PCR product was cloned into pCX-TOPO and transformed into One Shot TOPI OF' E. coli (Invitrogen). Transformants were selected on LB agar plates containing 15 ug/ml chloramphenicol and then analyzed for the presence and orientation of the insert by restriction enzyme analysis and diagnostic PCR using the M13R-KH90 primer set. Positive clones were then sequenced to confirm that the insert sequence was both error free and in-frame with respect to the CX leader and the C-terminal, truncated RsaA ORF. 91 Fusion Protein Expression, Purification and Analysis The rat-nca::RsaA-fusion protein construct, "KH89 to KH90 pCX-TOPO", was transformed into One Shot B5 BAC Caulobacter cells and transformants were selected for on PYE (in w/v: 0.2% peptone, 0.1% yeast extract, 0.02% MgS0 4 7H20,0.01% CaCl2 2H20) plates containing 2 ug/ml chloramphenicol and grown at 30°C. Several transformants were chosen for small scale expression experiments to confirm expression of the fusion protein prior to undertaking large scale production. Single colonies were inoculated into 5 ml PYE medium containing 2 ug/ml chloramphenicol and were grown overnight at 30°C. 4 ml of the overnight culture was then inoculated into 50 ml of M l 1 Expression Media (Invitrogen) and incubated at 30°C for 2 days with slow shaking (80-100 rpm). The fusion protein was in the form of an insoluble protein aggregate in the bacterial culture. In order to purify the fusion protein aggregate, the culture media was filtered through a nylon mesh that allowed the culture media to flow through but not the aggregate. To wash away any residual bacterial cells, the protein aggregate was transferred to a centrifuge tube and an equal volume of dH20 was added. The protein sample was centrifuged between 3,000 to 5,000 x g for just as long as it took to get up to speed and then stopped. This process would pellet the protein aggregate and not the bacteria and thus the two could be effectively separated. The water containing bacteria was then poured off and the aggregate was washed an additional two times. The protein aggregate was then dissolved in 8 M UREA/lOOmM Tris, pH 8.5 and stored at 4°C. To analyze the rat-NCA: :RsaA-fusion protein, 5 ul of the solubilized protein (at different dilutions) was mixed with 5 ul of 2X SDS-PAGE sample buffer and loaded on a 15% SDS-PAGE gel using established protocols (see below; Western Blotting) and the fusion protein was visualized using Coomassie blue staining. The RsaA protein is 32 kDa and the rat-NCA fusion fragment was 6 kDa giving a rat-NCA::RsaA fusion protein product of a predicted size of 38 kDa. 92 Generation of Polyclonal Antisera The generation of polyclonal antisera directed against the rat-NCA: :RSA fusion protein was performed by Covance Inc. Briefly, to generate polyclonal antisera, the rat-NCA: :RsaA fusion protein was first dialyzed into 6 M UREA/lOOmM Tris, pH 8.5. Subsequently, two New Zealand White female rabbits (BC188 and BC189) were injected with the fusion protein emulsified in Freund's complete adjuvant (250 ug protein/rabbit). Rabbits were boosted at 3-week intervals with fusion protein emulsified in Freund's incomplete adjuvant (first boost 125 ug and all subsequent boosts 100 ug protein/rabbit) a total of 5 times. Blood samples were taken 10 days post injection and a full body bleed after the last boost. Only the body bleeds were subjected to protein A purification. Antibody immunogenic response was monitored by ELISA and Western blots using the purified fusion protein and comparing pre-immune and immune serum. Western Blotting SDS-PAGE analysis was performed on protein samples denatured in 2X SDS-PAGE sample buffer. Proteins were resolved on various % acrylamide gels (Harlow and Lane, 1999) and protein sizes were determined by comparing to pre-stained molecular weight markers (Invitrogen). Protein quantification was carried out using the BCA Protein Assay Kit (Pierce). Gels were run at 60 V through the stacking gel and then at 120 V through the resolving gel in running buffer (25 mM Tris, 250 mM glycine pH 8.3, 0.1% SDS) using a mini-gel apparatus (BioRad). Proteins were transferred to Trans-Blot nitrocellulose membrane (BioRad) in a immersion transfer apparatus (BioRad) filled with transfer buffer (2 mM Tris, 192 mM glycine pH 8.3,20% methanol, 0.37% SDS) at 100 V for 1 hr. The membranes were then blocked overnight at 4°C in 5% milk powder, 5% BSA, PBS-T (PBS: 137 mM NaCI, 2.7 mM KC1,10 mM Na2HP04, 2 mM KH 2 P0 4 pH 7.5 T: 0.1% Tween 20). Membranes were then rinsed three 93 times for 15 min in PBS-T and incubated with primary antibodies (see below) in 5% milk powder, PBS-T for 1 hr at room temperature. Membranes were again washed three times for 15 min in PBS-T and incubated with horseradish peroxidase-labeled secondary antibodies (see below) in 5% milk powder, PBS-T for 1 hr at room temperature. Membranes were rinsed three times for 15 min in PBS-T and then incubated in ECL detection reagents for 1 min and exposed to film (Biomax MS double emulsion film, Kodak). For Western blotting, protein A purified anti-Rat: :NCA rabbit polyclonal antibodies (BC 188/189) were diluted (1:250 - 1:10,000) and the anti-FLAG M2 (Sigma) mouse monoclonal antibody was used at 10 ug/ml. The secondary antibodies, horseradish peroxidase labeled goat anti-rabbit and mouse IgG (Amersham) were diluted to 1:5,000. Immunofluorescent Staining HEK tsA201 cells were grown on poly-D-lysine coated 1 mm thick coverslips placed into 12 well culture dishes and were transfected with Lipofectamine (Invitrogen) according to the manufacturer's instructions. The HEK tsA201 cells were transfected with either full-length rat-nca A (F 10-9 pCDNA3), full-length xat-nca A with a C-terminal FLAG fusion (F 10-9::FLAG pCDNA3), or with pCDNA3. All transfections also included the plasmid pEGFP (Clontech) as a transfection control. The media was removed three days post-transfection and the coverslips were washed twice in PBS/20 mM glycine. The cells were then fixed in 3% para-formadehyde-PBS (pH 7.4-8.0) for 20 min at room temperature. The coverslips were then washed twice with PBS/20 mM glycine (5 min/wash) and cells were permeabilized with PBS/20 mM glycine/0.1% saponin (Sigma) for 20 min. The coverslips were then incubated with the primary antibodies (see below) in PBS/20 mM glycine/0.1% saponin for 60 min at room temperature. Coverslips were then rinsed in PBS/20 mM glycine/0.1% saponin and incubated with secondary antibodies (see below) in PBS/20 mM glycine/0.1% saponin for 60 min at room temperature. Coverslips were 94 then rinsed in PBS/20 mM glycine/0.1% saponin and incubated with DAPI in PBS/20 mM glycine/0.1% (1:10,000) for 5 min at room temperature. Coverslips were then rinsed in PBS/20 mM glycine/0.1% saponin, followed by a rinse in PBS and then dH 20 alone. Coverslips were allowed to air dry, mounted onto slides with 3 ul of mounting media (2.5% DABCO, 90% glycerol, PBS pH 8) and sealed with nail polish. The cellular expression pattern of the channels of interest were examined using a Zeiss fluorescent microscope. For immunofluorescent staining, anti-Rat: :NCA rabbit polyclonal antibodies (BC188/189) were diluted (1:250) and the anti-FLAG M2 (Sigma) mouse monoclonal antibody was used at 20 ug/ml. The secondary antibodies, donkey anti-mouse/rabbit Cy3 (Jackson Laboratories) were both diluted 1:250. Electrophysiological Recordings from HEK tsA201 Cells Functional analysis of the rat-NCA A channel was performed by heterologous expression in the HEK tsA201 cell line. HEK cells were grown in standard Dulbecco's modified Eagle's Medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum and 50 U/ml penicillin/streptomycin to 80% confluence. Cells were incubated at 37°C in a humidified atmosphere of 95% 0 2 and 5% C 0 2 and were enzymatically dissociated every 2-3 days with trypsin-EDTA (Invitrogen) and plated on 35 mm Petri dishes 12 hrs prior of transfection. Cells were transiently transfected using a standard calcium phosphate procedure. The rat-nca A cDNA was cotransfected with and without the Ca 2 + channel accessory subunits a25-l and Pn> (or p2a) at a molar ratio of 1:1:1; as well as with the CD8 antigen (2 ug) and pBluescript SK (15 ug) as carrier DNA. A total of 20 ug of cDNA was used in each transfection. Transiently transfected cells were identified visually by the binding of CD8-coated Dynabeads (Dynal) to CD8 expressing cells (Jurman et al., 1994). Control cells were transfected with pBluescript SK and the 95 Ca channel accessory subunits in order to examine the presence of endogenous ion currents in HEK cells under the experimental conditions chosen. These experimental protocols were also repeated with HEK cells transiently transfected with the Na + channel pi accessory subunit in place of the Ca 2 + channel accessory subunits. Microelectrodes were constructed from borosilicate glass (Sutter Instrument Co.) on a P-87 micropipette puller (Sutter Instrument Co.) and the tips of the recording electrodes were fire polished with a microforge (MF-83, Narishige). The resistance of the recording electrodes was between 4-6 MQ when filled with the internal solution and the bath was connected to ground by a 3 M KC1 agar bridge. Currents were recorded in the whole cell patch configuration at room temperature (20-24°C) using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Recordings were acquired at sampling rates of 2 or 5 kHz onto a personal computer equipped with pCLAMP software, version 6.03 (Axon Instruments). Functional expression of the rat-NCA A channel was evaluated 24-72 hrs after transfection by using different experimental protocols and different recording solutions with various compositions. In the first experimental protocol, the external recording solution contained (in mM): NaCI (116), KC1 (5), CaCl2 (1.8), MgCl 2 (1), HEPES (10) and glucose (5.5). The pH of the external recording solution was adjusted to 7.4 with NaOH. The internal recording solution contained (in mM): Kaspartate (105), EGTA (5), CaCl2 (5), MgCl 2 (1) and HEPES (10). The pH of the internal recording solution was adjusted to 7.2 with KOH. To measure whole cell currents the membrane potential was stepped from a holding potential of -90 mV to various test potentials ranging from -60 to +90 mV for 20 ms and then repolarized to -30 mV for approximately 50 ms. In the second experimental protocol, the external recording solution contained (in mM): NaCI (100), TEAC1 (35), HEPES (10), EGTA (2) and glucose (10). The pH of the external recording solution was adjusted to 7.4 with NaOH. In this experimental protocol, a 96 phosphorylation cocktail was included in the internal solution to avoid the potential wash out or run-down of the measured currents. The internal recording solution contained (in mM): CsCl (105), TEAC1 (25), CaCl2 (1), EGTA (11) and HEPES (10). The pH of the internal recording solution was adjusted to 7.2 with CsOH. Prior to experimentation, the phosphorylation cocktail consisting of 5 mM Mg-ATP, mycrocystin and phosphocreatin was added to the internal recording solution. To measure whole cell currents under this condition two different electrophysiological protocols were used. The first method consisted of voltage ramps from -100 mV to +100 mV and the second method consisted of 200 ms steps from -60 mV to +30 mV from a holding membrane potential of-120 mV. The electrophysiological experiments were performed by Dr. E. Garcia (T. Snutch Laboratory, University of British Columbia). 97 Chapter 3. Molecular Cloning and Expression of the Nematode and Mammalian nca Gene Family Background Considerable progress has been made towards identifying the different types of VGICs in native cells and their molecular counterparts, yet there still are a number of native ionic conductances that remain to be fully characterized. With the onset of genomic sequencing projects, novel VGICs can be identified using genome-based searches. The role that these "new" channels may play in normal physiological processes and disease states is not known. Thus, a complete study of these new channels is a necessary first step towards understanding their basic properties and contributions to neuronal physiology. In this study, I designed a strategy to identify novel four domain-type VGIC cti subunit genes by taking advantage of the sequence information contained in the C. elegans genome database. "Screening" of the C. elegans genome database with conserved rat HVA Ca 2 + channel ai subunit sequences lead to the identification of the nca genes in C. elegans. Additional screening of the GenBank EST databases with the C. elegans nca sequences lead to the identification of an nca homologue from rat. Using the sequence information gathered from the database screens, full-length cDNAs for nca-1, nca-2 and rat-nca were generated. Amino acid identity analyses between the predicted protein sequences of the NCA channels and that of representative Ca 2 + and Na+ channels showed that the nca family of VGICs are likely members 98 of a novel family of four domain-type VGICs that may have unique ion selectivity, activation and inactivation properties. Results Identification of the nca-1, nca-2 and rat-nca Genes In order to identify novel four domain-type VGICs, oligonucleotides were designed against structurally conserved regions present in the majority of rat HVA Ca 2 + channel oti subunits and were used to "screen" the C . elegans genome database. This "screen" was performed in early 1997 prior to the release of the entire C . elegans genome sequence ( C . elegans Sequencing Consortium, 1998) and the identification of the different ai subunit genes in C . elegans by Bargmann (1998). The screening strategy used in this study resulted in the identification of five potential four domain-type VGIC cti subunit genes in C . elegans. Three of the genes (unc-2, egl-19 and cca-1) were determined to represent homologues of mammalian Ca channels, while the other two genes (nca-1 and nca-2) appeared to represent a novel family of four domain-type VGICs. The unc-2 gene encodes a Ca 2 + channel ai subunit homologous to the mammalian non L-type channels (Schafer and Kenyon, 1995), while the egl-19 gene encodes a Ca channel ai subunit homologous to the mammalian L-type channels (Lee et al., 1997) and the cca-1 gene encodes a Ca 2 + channel ai subunit homologous to mammalian T-type channels (Bargmann, 1998; Cribbs et al., 1998; Perez-Reyes et al., 1998; Lee et al., 1999b; McRory et al., 2001). A rat homologue of nca-1 and nca-2 (called rat-nca) was identified by screening the GenBank EST and non-redundant data banks with the nca-1 and nca-2 gene sequences. To date, the corresponding native currents of the nca gene family have not been identified in either C. elegans or mammalian tissues. 99 The Structure of the nca-1 Gene The nca-1 gene spans -14.2 kb of genomic sequence and consists of 28 exons and 27 introns (Figure 12). The exons range in size from 65 bp (exon 2) to 844 bp (exon 19) with an average length of 208 bp. The introns range in size from 46 bp (intron 4) to 1004 bp (intron 23) with an average length of 312 bp. All introns in the nca-1 gene conform to the GT-AG splice consensus sequence. Exon 1 contains a putative start ATG methionine as determined by sequence analysis of clones "nca-1 5' RACE" and "B" and Exon 28 contains an in-frame stop codon (TAG) and a potential polyadenylation sequence (AATGAA) 115 bp downstream as determined from clone yk354d9. The AATGAA potential polyadenylation sequence is present in approximately 11% of all C. elegans cDNA clones examined (Blumenthal and Steward, 1997). In order to confirm the predicted intron-exon boundaries, several cDNAs were isolated, sequenced and compared to the nca-1 genomic sequence in WormBase (Figure 12). Two of these cDNAs, yk207g5 and yk354d9, were provided by Y. Kohara (C. elegans cDNA Project). The yk207g5 cDNA is -2.3 kb in length, extends from exon 3 to exon 15, encodes from the beginning of domain I S2 into the domain U-III linker, but does not contain exon 13. The yk354d9 cDNA is -3.2 kb in length, extends from exon 15 to the 3' end of the gene at exon 28, encodes from the domain II-ILT linker to the end of the carboxyl tail, but does not contain exon 13. The yk354d9 cDNA contains the stop codon followed by 132 bp of 3' UTR and a poly(A) tail. The polyadenylation consensus sequence is located 12 bp upstream of the poly(A) tail. Two additional cDNAs that overlapped with yk207g5 and yk354d9 were isolated either by PCR of a C .elegans yeast-two-hybrid library provided by R. Barstead (C. elegans Gene Knockout Project) or by RT-PCR of N2 total RNA. The first of these two additional overlapping clones, "N1-A3SDM", generated by PCR using the primer set KH107-KH-108 and a C. elegans yeast-two-hybrid library, is 1346 bp in length spanning from exon 10 to exon 19 and encodes from the domain II-IU linker to the beginning of domain III S6, but does not contain exon 13. 100 Figure 12. The Structure of the nca-1 Gene and Predicted Protein Products The nca-1 gene consists of 28 exons spanning -14.2 kb. Exons and introns are represented as boxes and lines, respectively. The nca-1 gene encodes for at least two proteins of 1797 aa (NCA-1) and 1831 aa (NCA-1 AS), respectively. The additional 102 bp of coding sequence in the "fixed N1-A4" clone and the extra 34 aa in the corresponding "NCA-1 AS" protein are represented as triangles on the diagram. Similar to other four domain-type VGIC ci(i) subunits, NCA-1 possesses four homologous domains (I-IV), each consisting of six putative hydrophobic membrane-spanning segments. The transmembrane segments are indicated as boxes and the cytoplasmic and extracellular domains are indicated as lines. Shown below the protein are the overlapping cDNA clones that together contain the entire coding sequence of nca-1 and were used to determine intron-exon boundaries. 101 102 The second of these two overlapping clones, "fixed N1-A4" is 1408 bp in length spanning from exon 10 to exon 19 and encodes from the domain II-III linker to the beginning of domain III S6 and contains exon 13. An additional cDNA clone representing the remaining 267 bp of the nca-1 coding sequence was isolated by RT-PCR using N2 total RNA and the primer KH108 for first strand cDNA synthesis and the KH106-KH117 primer set for PCR amplification. The resulting cDNA (clone "B") is 877 bp in length, overlaps with clone yk207g5, spans from exon 1 to exon 7 and encodes from the amino terminal methionine to the pore-forming loop (P-Loop) of domain I. Analysis of clones yk207g5, yk354d9, "N1-A3SDM" and "fixed N1-A4" suggests that the nca-1 gene undergoes alternative splicing of exon 13. Inclusion of exon 13 results in a 102 bp insertion in the nca-1 RNA transcript and a corresponding 34 aa insertion in the domain 11-111 linker of the NCA-1 protein. Consequently, alternative splicing of the nca-1 gene results in at least two different NCA-1 protein products, the shorter 1797 aa "NCA-1" protein (AY555271) and the longer 1831 aa "NCA-1 AS" protein, with predicted molecular weights of 212 and 208 kDa, respectively (Figure 12). Comparison of the Gene Structure oi nca-1 with that Predicted in WormBase A detailed comparison was made between the predicted intron-exon boundaries for the nca-1 gene deduced from the cDNA clones isolated in this study and that predicted for CI 1D2.6 in WormBase at the time the nca-1 gene was identified in this study (AF045640). Analysis of the gene structure of nca-1 revealed a major discrepancy between the number of exons present in both predictions of the nca-1 gene. The sequence annotation of CI 1D2.6 in WormBase predicted that the nca-1 gene consisted of only 23 exons effectively spanning from exon 1 to 22 of the prediction made in this study and encodes for a predicted protein of only 1581 aa. This dramatically shortened protein is due to the effective elimination of the carboxyl terminus of the 103 putative NCA-1 protein predicted in WormBase (Figure 12). In contrast, the analysis performed in this study suggested that the nca-1 gene consists of 28 exons and represents the entire coding region of the nca-1 gene, as well as the 5' and 3' UTRs. Furthermore, the CI 1D2.6 prediction in WormBase placed additional exons between exon 7 and 8 and before exon 1 of the prediction made in this study and did not include the alternatively spliced exon 13 (Figure 12). Analysis of clone yk354d9 revealed the presence of six additional exons (exons 23 to 28) beyond the 3' end of that reported in WormBase for CI 1D2.6. By using the splice site 65 bp upstream of the predicted stop site in exon 22 of CI 1D2.6 and the six additional exons, the prediction made in this study removed 21 aa from CI 1D2.6 and then extended the open reading an additional 348 aa and now encodes a carboxyl terminus. In addition, analysis of clone yk207g5 revealed a number of other discrepancies between the two predictions. CI 1D2.6 has an additional 54 bp exon between exons 7 and 8 of the prediction made in this study and used different putative splice sites to extend the 3' end of exon 7 by 64 bp and to extend the 5' end of exon 8 by 20 bp. Furthermore, CI 1D2.6 used a different splice site at the 3' end of exon 10, extending this exon by 21 bp. Clone yk207g5 did not contain the additional 54 bp exon and used different splice sites to generate shorter sized exons 7, 8 and 10. Consequently, the prediction made in this study removed 53 aa from this region in CI 1D2.6. Furthermore, analysis of clone "nca-1 5' RACE" revealed a discrepancy in the determination of exon 1, as CI 1D2.6 has an extra exon at the 5' end as compared to the prediction made in this study. Exon 1 in the prediction made in this study is 298 bp in length and contains 171 bp of 5' UTR and 127 bp of coding sequence. Exon 1 in CI 1D2.6 is 145 bp in length with the start site at bp 1. The first intron in the CI 1D2.6 is 121 bp and the second exon is 156 bp in length. According to the CI 1D2.6 prediction, the 3' end of exon 1 as predicted in this study is the same as the 3' end of exon 2 of CI 1D2.6, but the predicted start site of the nca-1 gene in this study occurs within this exon, 126 bp upstream and the predicted 5 'UTR of the nca-104 1 gene predicted in this study extends an additional 171 bp upstream right through the first intron of CI 1D2.6 and stops 21 bp into exon 1 of CI 1D2.6. Thus, the prediction made in this study removes an additional 58 aa from the open reading frame and now encodes the predicted 1797 aa "NCA-1" protein (AY555271). The Predicted NCA-1 Protein Products The predicted protein sequence of the NCA-1 protein products are shown in Figure 13. The primary sequence of the NCA-1 proteins are similar to that of other cloned four domain-type VGICs. Like other four domain-type VGIC ct(i) subunits, the NCA-1 proteins are predicted to consist of four homologous, mainly hydrophobic domains (I-IV), each consisting of six putative membrane-spanning segments (S1-S6) and a pore-forming loop (P-Loop). There are 40 consensus sites for phosphorylation by PKA, PKC and calmodulin-dependent protein kinase in predicted cytoplasmic regions in the NCA-1 AS protein (Bairoch et al., 1997; Kreegipuu et al., 1999). Of the 40 potential phosphorylation sites, three lie within the extra 34 aa in the domain II-III linker of NCA-1 AS. The three extra putative phosphorylation sites present in the alternatively spliced NCA-1 AS channel may allow for the differential modulation of this channel isoform. There are two N-glycosylation consensus sites (N-X-S/T) in the NCA-1 proteins in predicted extracellular regions (Bairoch et al., 1997; Kreegipuu et al., 1999). The Structure of the nca-2 Gene The nca-2 gene spans -14.2 kb of genomic sequence and consists of 23 exons and 22 introns (Figure 14). The exons range in size from 113 bp (exon 3) to 514 bp (exon 16) with an average length of 244 bp. The variation in intron size is even more pronounced. The majority of introns in nca-2 range between 48 bp (intron 8) and 463 bp (intron 6), however introns la and lb are 4522 bp and 917 bp, respectively. All introns in the nca-2 gene conform to the GT-AG 105 Figure 13. The Complete Amino Acid Sequence of the NCA-1 ai Subunit Transmembrane segments are indicated with lines above the sequence. Potential N-linked glycosylation sites are indicated with (+) and putative phosphorylation sites are indicated with the following symbols: $ = cAMP-dependent protein kinase; # = cAMP-dependent protein kinase and calmodulin-dependent protein kinase JJ; • = protein kinase C; * = cAMP-dependent protein kinase, calmodulin-dependent protein kinase II and protein kinase C. The 34 amino acids in red indicate the alternatively spliced sequence in the NCA-1 AS protein and the amino acids in bold (E and K) indicate the coordinating glutamate and lysine residues that may contribute to ion selectivity. 106 #• $ $ • I SI MLJ^lKNSSSRGAPGSAAGFGARESI^ISDMLSSQHKKPVRSSyVESDRVEW^KIACTI 60 I 52 # • SMITVCIHTPRTIELFQPLNY11LAADFISVSIFMLDSVLRIHYEGIFRCDS SYLSNRWS 120 I S3 I S4 QFSVFIS11HLLSFLLHCYQLIDNFFPFLHLNYRAWYGAIRSIRPFI11RLIPLWKFKL 180 $ I S5 + PKNRIEQLLKRSSQQVKNVTLFFVFFMTLYAIFGIQLFGRMDYHCVQPKTDPNNVTIMDL 24 0 I P-LOOP AIPDTMCAPEGIGGYECPAPMVCMQLNLNAKGEGFYGMFNDFGASVFTVYLAASEEGWVY 300 I S6 VLYDCMDSLPSYLAFLYFCTLIFFLAWLVKNVFIAVITETFAEIRVQFSEMWQKKEVTLD 360 II SI EGFRKKLEKTDDGWRLIRLDGEVEPEGPKQKLQWMLRSMYFQCFVIIFWINAIGNAMFV 420 II S2 II S3 YRHDETDKPRKYNFYLFEVGFTILFNVECIIKILCYGFRNFIRRGIFKFELILCLGSSLN 480 II S4 II S5 CVKFFYERNYFTYFQTFRLLRLIKASPILEDFVWKIFSPGKKLGGLVIFTIAFICCYSAI 540 II P-LOOP SLQLFYSVPNLHHFRTFPQAFMSMFQIITQEGWTDFWEVLRATDDNLVPLVALYFVAYH 600 II S6 # • $ LFVTLIVLSLFVAVILDNLEMDEELKKVKQLRAREATTSMRSTLPWRLRVFEKFPTRPQM 660 $ # • AAMRKADSDFPMPKVRGSFTHQFAVDHSLETTDVIESDFEFPKRIMKSAGKRKISKSGLT 720 • $ • $ $ * FRQIGSTSLRCSLNNLLEMSDRTRQSLSNSLSFLPHFTRSSGSLYPRKDALPKSRSMTGK 780 $ FLQTAVRNKQFNMYSENGDLSRPSDSAPKKNAKQGEIDIRALQQKRQLAEITRNRIEEDM 840 $ • RENHPFFDRPLFLVGRASQLREFCKKMVHSKYDSQDDGTNGGAKTKKRFKEIRALIGIMP 900 III SI III S2 YIDWAMATVTIVSCISMLFESPWPTTGENLVMNNGYLQISDYFFVLSMTFELCVKIIANG 960 • III S3 III S4 LFFTPKAWRDVGGVWLFIYFTSVIFLAWMPKHVEINSLAQFLMICRAMRPLRIYTLVP 1020 1 0 7 I I I S5 + H I R R W L E FFRG F K E I L L V T I L M I W M F I FAS FGVQIVGGKLAACNDPTVS SRENCTGVF 1080 I I I P-LOOP WQKLEVTRLEVYGKDTEAMHPKIiyrVPRWTNPRNFNFDHVGNAMLALFETLSFKGWNVIR 114 0 I I I S6 $ DILWSRHGPWAVVFIHIYVFIGCMIGLTLFVGWIANYTQNRGTALLTVDQRRWHDLKAR 1200 • # I V SI LKMAQPLHVPPKPSESARLRTKLYDLTMSRWFNQAFALLWLNSFTLVIPWNVEEEEQRA 12 60 IV S2 • I V S3 TYVFTVTALAAFMNMLFVIEIILKVIAYTFSGFWQSRRNRVDLLITVFGVIWIFLHFFVA 1320 IV S4 LPSSKIDVDVQVELKKFTYTFGYLWILRFFTIASRNSTLKMLMLTVIMSMFRSFFIITA 1380 IV S5 I V P-LOOP LFLLVLFYAYTGVILFPMVKYGMAVSKHVNFRTASEALWLFRCLTGEDWNDIMHDCMRS 14 4 0 I V S6 APFCYWNEGMNYWETDCGNFYGAIIYFCSFYLIITYIVRNLLVAVIMENFSLFYSSEEDA 1500 LLSYADIRNFQYVWNMVDQEQKRTIPVERVKFLLRLLKGRLEVDPEKDRILFKHMCYEME 1560 • $ • RLHNGEEVSFHDVLYMLSYRSVDIRKSLQLEELLQREELEFIIEEEVAKQTIRTWLEGCL 1620 RKMRNPSQKDAEGVLPFGGGHPVIHSSGHSSISHEETVAQRLRFENIRGDSVDTEETESS 1680 EEETPPPIRKKAAVKNRRGSIPDVLSRTGLFQEAARKFMVGSSSEKKQVKSRSPETVQLL 17 40 # # • PKRANSEIRKGSGQPKNFHLQLNVYDLPDVEERGEDSPFSPKNLSDDFNGEHSPLVITPS 1800 LPVPPTHGSPRPLMPCETTKDIEKWWNSLVD 1831 108 Figure 14. The Structure of the nca-2 Gene and Predicted Protein Products The nca-2 gene consists of 23 exons spanning -14.2 kb. Exons and introns are represented as boxes and lines, respectively. The nca-2 gene encodes for at least two proteins of 1763 aa (NCA-2) and 1785 aa (A5' NCA-2), respectively. The additional 66 bp of coding sequence in the "Ul" clone and the extra 22 aa in the corresponding "A5' NCA-2" protein are represented as red boxes on the diagram. Similar to NCA-1 and other four domain-type VGIC ct(i) subunits, NCA-2 has four homologous domains (I-TV), each consisting of six putative hydrophobic membrane-spanning segments. The transmembrane segments are indicated as boxes and the cytoplasmic and extracellular domains are indicated as lines. Shown below the protein are the overlapping cDNA clones that together contain the entire coding sequence of nca-2 and were used to determine intron-exon boundaries. 109 110 splice consensus sequence. Exon la and lb both contain putative start ATG methionines as determined from sequence analysis of cDNA clones "Ul" and "fixed LI" and exon 22 contains an in-frame stop codon (TAA) and a potential polyadenylation sequence (AATAAA) 134 bp downstream as determined from clone yk24d9. The AATAAA potential polyadenylation sequence is present in approximately 56% of all C. elegans cDNA clones examined (Blumenthal and Steward, 1997). In order to confirm the predicted intron-exon boundaries, several cDNAs were isolated, sequenced and compared to the nca-2 genomic sequence in WormBase (Figure 14). The first of these cDNAs, yk24d9, was provided by Y. Kohara (C. elegans cDNA Project). The yk24d9 cDNA is -3.8 kb in length, extends from the very end of exon 7 to the 3' end of the gene at exon 22 and encodes from the pore-forming loop (P-Loop) of domain II to the end of the carboxyl tail. The yk24d9 cDNA contains the stop codon followed by 154 bp of 3' UTR and a poly(A) tail. The polyadenylation consensus sequence is located 15 bp upstream of the poly(A) tail. Three overlapping cDNA clones representing the remaining -1.8 kb of the nca-2 coding sequence were isolated either by RT-PCR of N2 total RNA or by PCR of a C. elegans yeast-two-hybrid library provided by R. Barstead (C. elegans Gene Knockout Project). The clones "Ul" and "fixed LI" were amplified by RT-PCR using N2 total RNA and an oligo dT primer for first strand cDNA synthesis and the SL1-KH82AOS primer set for PCR amplification. The larger of these two clones, "Ul", is 888 bp in length spanning from exon la to exon 4 and encodes from the upstream amino terminal methionine to the beginning of the pore-forming loop (P-Loop) of domain I. The smaller of these two clones, "fixed LI", is 770 bp in length, spans from exon lb to exons 4 and encodes from the downstream amino terminal methionine to the beginning of the pore-forming loop (P-Loop) of domain I. Analysis of clones "Ul" and "fixed LI" suggests that the nca-2 gene is SL1 /raw-spliced and may be alternatively transcribed. Exon la and lb are both preceded by a SL1 £ra>w-splice consensus sequence (AG) (Blumenthal and Steward, 1997) 111 and in exon la, the start site is 91 bp downstream of the SL1 trans-splice site, whereas in exon lb the start site is 49 bp downstream of the SL1 /ram-splice site. Inclusion of exon la results in a 66 bp insertion in the coding region of nca-2 RNA transcripts and a corresponding 22 aa addition to the amino terminus of the NCA-2 protein. The third overlapping cDNA, "115-116 PCR" was generated by PCR using the primer set KH115-KH116 and a C. elegans yeast-two-hybrid library and overlaps "Ul" and "fixed LI" at the 5' end and yk24d9 at the 3' end. The "115-116 PCR" clone is -1.9 kb in length, spans from exon 2 to exon 9 and encodes from domain I SI to the beginning of the domain 11-111 linker. Analysis of clones "Ul" and "fixed LI" suggests that the nca-2 gene undergoes alternative splicing of exon la. Consequently, alternative transcription/splicing of the nca-2 gene results in at least two different NCA-2 protein products, the shorter 1763 aa "NCA-2" protein and the longer 1785 aa "A5' NCA-2" protein (AY555272) with predicted molecular weights of 206 and 204 kDa, respectively (Figure 14). Comparison of the Gene Structure of nca-2 with that Predicted in WormBase A detailed comparison was made between the predicted intron-exon boundaries for the nca-2 gene deduced from the cDNA clones isolated in this study and that predicted for C27F2.3 in WormBase at the time the nca-2 gene was identified in this study (U40419). Analysis of the gene structure of nca-2 revealed a major discrepancy between the number of exons present in the predictions of the nca-2 gene. The sequence annotation of C27F2.3 in WormBase predicted that the nca-2 gene consisted of only 17 exons spanning from exon 2 to 18 of the prediction made in this study and encodes for a predicted protein of only 1410 aa. This dramatically shortened protein is due to the effective elimination of both the amino and carboxyl termini of the putative NCA-2 protein predicted in WormBase (Figure 14). In contrast, the analysis of the nca-2 gene performed in this study suggested that the nca-2 gene consists of 23 exons and represents the 112 entire coding region of the nca-2 gene, as well as the 5' and 3' UTRs. Analysis of clone yk24d9 revealed the presence of four additional exons (exons 19 to 22) beyond the 3' end of that reported in WormBase for C27F2.3. By using the splice site 17 bp upstream of the predicted stop site in exon 17 of C27F2.3 and the four additional exons, the prediction made in this study removed the last 5 aa from C27F2.3 and then extended the open reading an additional 282 aa and now encodes a carboxyl terminus. Furthermore, analysis of clone "Ul" revealed the presence of two additional exons (exons la and lb) beyond the 5' end of that reported in WormBase for C27F2.3. By using the splice site 95 bp upstream of the predicted start site in exon 2 of C27F2.3 and the two additional exons, the prediction made in this study extended the open reading frame an additional 98 aa and now encodes an amino terminus and the predicted 1785 aa "A5' NCA-2" protein (AY555272). The Predicted N C A - 2 Protein Products The predicted protein sequence of the NCA-2 protein products are shown in Figure 15. The primary sequence of the NCA-2 proteins are similar to that of other cloned four domain-type VGICs. Similar to NCA-1 and other four domain-type VGIC a(i) subunits, the NCA-2 proteins are predicted to consist of four homologous, mainly hydrophobic domains (I-IV), each consisting of six putative membrane-spanning segments (S1-S6) and a pore-forming loop (P-Loop). There are 42 consensus sites for phosphorylation by PKA, PKC and calmodulin-dependent protein kinase in predicted cytoplasmic regions in the A5' NCA-2 protein (Bairoch et al., 1997; Kreegipuu et al., 1999). Of the 42 potential phosphorylation sites, one lies within the extra 22 aa at the amino terminal of the A5' NCA-2 protein. The extra putative phosphorylation site present in the alternatively spliced A5' NCA-2 channel may allow for the differential modulation of this channel isoform. There are three N-glycosylation consensus sites (N-X-S/T) in the NCA-2 proteins in predicted extracellular regions (Bairoch et al., 1997; Kreegipuu et al., 1999). 113 Figure 15. The Complete Amino Acid Sequence of the NCA-2 ai Subunit Transmembrane segments are indicated with lines above the sequence. Potential N-linked glycosylation sites are indicated with (+) and putative phosphorylation sites are indicated with the following symbols: $ = cAMP-dependent protein kinase; • = protein kinase C; ~ = cAMP-dependent protein kinase and protein kinase C; # = cAMP-dependent protein kinase and calmodulin-dependent protein kinase II; * = cAMP-dependent protein kinase, calmodulin-dependent protein kinase II and protein kinase C. The 22 amino acids in red indicate the alternatively spliced sequence in the A 5 ' NCA-2 protein and the amino acids in bold (E and K ) indicate the coordinating glutamate and lysine residues implicated in ion selectivity. 114 • • # • # MTSTTAKLLGLSACRAAALSTTMLTRRKSSIDGTKTERNRRRGESIGGAFTDMINIEPTS 60 I S I I S2 I INKSTELLHERFLRDMVRVACLLSMISLCLHTPETIKMWPPLNYIILANDVIVTLIFIG 120 I S3 EAAVTINQNGLFDNQNSYLRDRWYQFEFFLLINHILSCVIHIYELCSIWFPALNFVYYPW 180 I S4 $ I S5 LGALRSARPFIFLRFIRSIVRFKLPKNRIKLIIKRSSQQIQNVTIFFMFFVFSYAIMGVQ 24 0 + + LFGRLNYHCWNGTDPNNVTIADLAIPDTMCSQKGAGGYECPGNMVCMRLQLKPQEEGFY 300 I P-LOOP I S6 GQFSDFASSLFTWLAASQEGWWLYDCLDSLPSFLAFFYFVTLIFFLAWLVKNVFIAV 360 # • # ITETFAEIRVQFSEMWQTREATTDHVYTQKLEKDEDGWKLVEVDKYNRAHSNNSLFLHTI 420 I I S I I I S2 VTSTAFQTVMQLLILANAIFHATFVFYHDESDQIRKIWYYYVEVGFTILFNTEVIIKIYA 480 I I S3 I I S4 FGWKAYIARGQHKFDCILCVGSSLNAIWVLYETNIFTYFQVFRIARLIKASPMLEDFVYK 54 0 I I S5 I I P-LOOP IFGPGKKLGGLVIFTGILLIVTSAISLQLFCYVPKLNKFTNFAVAFMSMFQIITQEGWTD 600 I I S6 WIEILRACNEQAVPFVAIYFVAYHLLVTLFVLSLFVAVILDNLEMDEELKKVKQLKARE 660 • # • $ QDTIKTTLPMRLRIFNRFPTAPTMVTMKKVSSEFPLPKIRDSFTRQFADEFVETSDDTIQ 720 • # #• • • EIGFKVRSMLSGRGPSKETRITTTIRHVGQLSNKTILTSMLTESNRNRALFSESNQHLAS 780 #• # • • LTRSNTSSKHGKSGALSTNSRSRTRGLASLKGKHMVEGFKENGDLRPEDTARKVEKHGEI 84 0 $ DFKALQMKRAHAEITRNRIEEEMRENHPMFDRPLFLVGRESSLRRMCQLIAHSRHSYDQN 900 * I I I S I DGQHRKHSNKYKQFHDFLAIMTYMDWTMVLVTTLSCCSMLWESPWPTTGENLIFNNFYLQ 960 I I I S2 • I I I S3 IAEYIFVLV14SFELIVKCIANGLFFTPKALVTDVGDILTIFIYITSLMFLIWMPNHIEIN 1020 115 I I I S4 I I I S5 S WAQLLMVCRAMRPLRVYALI PH I R R V W E L C R G F R E I L L V T I L L W L M F I FAS FGVQLV 1080 + GGKLASCNDPMITSRENCTGLYDVKLFVTRMEVYGKNDNLMHPSIIVPRVWTNPRNFNFD 114 0 I I I P-LOOP I I I S6 HIGNAMIJ\LFETLSYKGWNWRDVLYLRHGAWAVLFIHIYVFIGCMIGLTLFVGWVANY 1200 TENRGTALLTVDQRRWHDLKARLKMAQPLHVPPKPPESAKLRCYLYDLTTSRWFKQLFAV 12 60 IV SI IV S2 • LVVWSFTLVIPWNVSEEQDRKTFLLCLTVISAICNILFTLECLLKMIAFTLSGFWQSRR 1320 IV S3 I V S4 NRIDFIITILGINWIVFHFLFQLPAYFAGGITEWKRLTYTYGYLWILRFFTIAGRKSTL 1380 . I V S5 KMLMLTWMSMVRSSFIIAAMFLLVLFYANAGWLFGMVKYGQAVGKHVNFRNGREALW 14 4 0 I V P-LOOP I V S6 LFRSVTGEDWNDIMHDCMRAPPFCNWHPGLSYWQTDCGNYVGAIVYFCSFYLIITYIVLN 1500 LLVAIIMENFSLFYS SEEDALLSYADIRNFQLVWNMVDIEQKRSIPVRRVKFLLRLLKGR 1560 • $ LEVNDESDGLLFKHMCHEMERLHNGDDVSFHDVLNMLSYRSVDIRKSLQLEELLQREELE 1620 YIIEEEVAKHTIRAWLENCLKNIKAKQNNTLGKMS SIGSTFAFPQSQEVLTKGWLTEAS 1680 • # • * • ~ PEEDSLQGDKGSGKKKAQRGNSITEIVAEAQKKSVKRATDKISERRGTLRQMQMGTYDEL 174 0 # EEVEEDE DT DGEIRRS S FEYSGVDMIQMS HEKQLEDVKTWWTLC D 1785 116 Cloning and Sequencing of rat-nca Mammalian homologues of nca-1 and nca-2 were identified by screening the GenBank EST and non-redundant data banks with the genomic sequences of nca-1 and nca-2. Three resulting ESTs (AA157945, AA683293 and AA967995) and one rat cDNA (AF078779) were judged to encode novel NCA-like proteins. Utilizing these sequences, primers were generated against domain III S2 (KH16) and domain ITI S6 (KH15) of the rat-nca cDNA sequence in the database. The clone "KH15-KH16" was obtained by RT-PCR using rat brain total RNA, the primer KH15 for first strand cDNA synthesis and the KH15-KH16 primer set for PCR amplification. The 646 bp product, spanning from bp 2883-3528, was then used to screen a rat brain cDNA library. After four rounds of plaque purification, three positives cDNA clones ("10-2111", "9-1 111" and "1-2111") were isolated and sequenced. All three cDNA clones encoded for rat-nca and cDNA clone "10-2111" was used as a template to piece together a full-length rat-nca A cDNA (5229 bp). In order to verify the putative start site of the rat-nca A cDNA, 5' RACE was performed using rat brain total RNA. Primer KH47A was used to reverse transcribe the RNA. The nested primer KH85 was used in the subsequent PCR steps. The furthest upstream primer, KH73, was used as a probe during Southern blotting. The single positive product was subcloned into the pGEM-T Easy vector and sequenced. From the cDNA clones "5' RACE rat-nca" and "10-2111" it was determined that the first start site (ATG) in the rat-nca open reading frame is at bp 78 in clone "10-2111" and is preceded by an in-frame stop codon (TGA) at bp 12 in the 5' UTR. The flanking DNA sequence of the putative start site TCACCATGC is similar to the consensus initiation sequence CCA/GCCATGG identified from mRNAs of diverse eukaryotic cells (Kozak, 1984). Examination of the open reading frame revealed a stop codon at bp 5292 leading to a predicted rat-NCA A protein of 1738 aa (AY555273). 117 The rat-nca cDNA clone, "U2" was obtained by RT-PCR using rat brain total RNA, the primer KH83 for first strand cDNA synthesis and the upstream KH61-KH84 primer set (bp 4785-5208 of rat-nca A cDNA) for PCR amplification. Analysis showed that cDNA clone "U2" contained a 96 bp insertion at bp 5101 of the rat-nca A cDNA with the resulting rat-NCA B protein having an additional 32 aa in its carboxyl tail (total of 1770 aa). Thus, the rat-nca gene encodes for at least two different alternatively spliced protein products of 1738 (rat-NCA A) and 1770 aa (rat-NCA B) with predicted molecular weights of 200 and 204 kDa, respectively. The predicted protein sequence of the rat-NCA protein products are shown in Figure 16. The primary sequence of the rat-NCA proteins are similar to that of the C. elegans NCA-1 and NCA-2 channels and other cloned four domain-type VGIC ct(i) subunits. There are 29 consensus sites for phosphorylation by PKA, PKC and calmodulin-dependent protein kinase in predicted cytoplasmic regions in the rat-NCA proteins. In addition, there are three N-glycosylation consensus sites (N-X-S/T) in the rat-NCA proteins in predicted extracellular regions (Bairoch et al., 1997; Kreegipuu et al., 1999). Sequence Analysis of Rat-nca A At roughly the same time that the full-length rat-nca A cDNA was generated in this study, another group using a similar strategy screened GenBank and identified the nca family in C. elegans and ultimately cloned the same rat nca homologue which they called Rb21 (Lee et al., 1999a). A detailed comparison was made between the nucleotide sequence of rat-nca A (AY555273), that reported for Rb21 (AF078779) and that predicted from the working draft of the rat genome project (NP 705894). This comparison revealed that the 1738 aa protein predicted from the rat-nca A cDNA is the same as that predicted in the rat genome database but is one amino acid longer than that predicted for the Rb21 protein (1737 aa). Comparison of the Rb21 cDNA with Rat-nca A and that predicted in the rat genome database revealed that the 118 Figure 16. The Complete Amino Acid Sequence Of The rat-NCA ai Subunit Transmembrane segments are indicated with lines above the sequence. Potential N-linked glycosylation sites are indicated with (+) and putative phosphorylation sites are indicated with the following symbols: $ = cAMP-dependent protein kinase; • = protein kinase C; ~ = cAMP-dependent protein kinase and protein kinase C; # = cAMP-dependent protein kinase and calmodulin-dependent protein kinase II; * = cAMP-dependent protein kinase, calmodulin-dependent protein kinase II and protein kinase C. The 32 amino acids in red indicate the alternatively spliced sequence in the rat-NCA B protein and the amino acids in bold (E and K) indicate the coordinating glutamate and lysine residues responsible for ion selectivity. 119 * I S I MLKRKQSSRVEAQPVTDFGPDESLSDNADILWINKPWVHSLLRICAIISVIPVCMNTPMT 60 I S2 I S3 FEHYPPLQYVTFTLDTLLMFLYTAEMIAKMHIRGIVKGDSSYVKDRWCVFDGFMVFCLWV 120 I S4 $ SLVLQVFEIADIVDQMSPWGMLRIPRPLIMIRAFRIYFRFELPRTRITNILKRSGEQIWS 180 I S5 + + VSIFLLFFLLLYGILGVQMFGTFTYHCWNDTKPGNVTWNSLAIPDTHCSPELEEGYQCP 240 I P-LOOP PGFKCMDLEDLGLSRQELGYSGFNEIGTSIFTVYEASSQEGWVFLMYRAIDSFPRWRSYF 300 I S6 # YFITLIFFIAWLVKWFIAVIIETFAEIRVQFQQMWGTRSSTTSTATTQMFHEDAAGGWQ 360 ' I I S I LVAVDWKPQGRAPACLQKMMRSSVFHMFILSMVTVDVIVAASNYYKGENFRRQYDEFYL 420 I I S2 I I S3 AEVAFTVLFDLEALLKIWCLGFTGYISSSLHKFELLLVIGTTLHVYPDLYHSQFTYFQVL 4 80 I I S4 I I S5 RWRLIKISPALEDFVYKIFGPGKKLGSLWFTASLLIVMSAISLQMFCFVEELDRFTTF 54 0 I I P-LOOP I I S6 PRAFMSMFQILTQEGWVDVMDQTLNAVGHMWAPLVAIYFILYHLFATLILLSLFVAVILD 600 NLELDEDLKKLKQLKQSEANADTKEKLPLRLRIFEKFPNRPQMVKISKLPSDFTVPKIRE 660 $ $ $ • SFMKQFIDRQQQDTCCLFRILPSTSSSSCDNPKRPTVEDNKYIDQKLRKSVFSIRARNLL 720 # # # • $ EKETAVTKILRACTRQRMLSGSFEGQPTKERSILSVQHHIRQERRSLRHGSNSQRISRGK 7 80 # • # SLETLTQDHSNTVRYRNAQREDSEIKMIQEKKEQAEMKRKVQEEELRENHPYFDKPLFIV 840 I I I S I GREHRFRNFCRVWRARFNASKTDPVTGAVKNTKYHQLYDLLGLVTYLDWVMITVTICSC 900 I I I S2 ISMMFE S PFRRVMHAPTLQIAEYVFVIFMSIELNLKIMADGLFFTPTAVIRDFGGVMDIF 960 I I I S3 I I I S4 IYLVSLIFLCWMPQNVPAESGAQLLMVLRCLRPLRIFKLVPQMRKWRELFSGFKEIFLV 1020 120 I I I S5 + SILLLTLMLVFAS FGVQLFAGKLAKCNDPNIIRREDCNGIFRINVSVSKNLNLKLRPGEK 1080 I I I P-LOOP KPGFWPRWANPRNFNFDWGNAMLALFEVLSLKGWVEVRDVIIHRVGPIHGIYIHVFV 1140 I I I S6 FLGCMIGLTLFVGWIANFNENKGTALLTVDQRRWEDLKSRLKIAQPLHLPPRPDNDGFR 1200 I V S I IV S2 AKMYDITQHPFFKRTIALLVLAQSVLLSVP<WDVEDPVTVPLATMSVVFTFIFVLEVTMKI 1260 • IV S3 IV S4 IAMSPAGFWQSRRNRYDLLVTSLGVVWWLHFALLNAYTYMMGACVIVFRFFSICGKHVT 1320 I V S5 LKMLLLTVWSMYKSFFIIVGMFLLLLCYAFAGWLFGTVKYGENINRHANFSSAGKAIT 1380 IV P-LOOP IV S6 VL FRIVTGEDWNKIMHDCMVQPP FCT PDE FTYWATDCGNYAGALMYFC S F Y V I I A Y I M L N 14 40 LLVAIIVENFSLFYSTEEDQLLSYNDLRHFQIIWNMVDDKREGVIPTFRVKFLLRLLRGR 1500 • $ LEVDLDKDKLLFKHMCYEMERLHNGGDVTFHDVLSMLSYRSVDIRKSLQLEELLAREQLE 1560 • • $ YTIEEEVAKQTIRMWLKKCLKRIRAKQQQSCSIIHSLRESQQQELSRFLNPPSIETTQPS 1620 • # • # EDTNANSQDHNTQPESSSQQQLLSPTLSDRGGSRQDAADTGKPQRKIGQWRLPSAAIAEK 1680 TAPVNKMSSYSQDPEREEEEVSKGFLAPKPISHSVSSVNLRFGGRTTMKSWCPCMNPMPD 1740 # TASCGSEVKKWWTRQLTVESDESGDDLLDI 1770 121 Rb21 cDNA contains a total of four basepair differences. The first of these basepair changes is a silent A1478T transition (numbered according to the rat-nca A cDNA), whereas the other three basepair changes are nucleotide deletions that alter the reading frame at the beginning of the carboxyl tail. The Rb21 cDNA does not contain base pairs A4517, G4525 and C4535 which, as a consequence, changes the amino acid sequence from the six amino acid sequence KREGVT (aa 1480 to 1485) present in the rat-NCA A protein and that in the rat genome database to the five amino acid sequence NERCD present in the Rb21 protein. Comparison of the rat-nca A cDNA with that predicted in the rat genome database revealed three basepair differences. The first of these changes was a T231C transition that results in a S52P amino acid change in the domain I SI region of the rat-NCA A protein. Comparison of this region with the predicted protein sequences of NCA-1 and NCA-2 revealed that this amino acid is conserved in NCA family proteins as NCA-1 contains a threonine (T) and NCA-2 contains a serine (S) at this position in the domain I SI region (Figure 17). The second change found in the predicted protein sequence of rat-NCA A was a silent G2319A transition, while the third change was a G1850A transition that results in a A748T amino acid change in the domain H-in linker. Comparison of the Primary Structure of NCA-1, NCA-2 and rat-NCA NCA-l/NCA-2 To compare the deduced protein sequences of NCA-1 and NCA-2, all of the different regions of the channels were aligned separately using the CLUSTAL W algorithm (Higgins and Sharp, 1988) and the percent identities and similarities were determined (Table 4). The similarity analysis showed that the highest degree of sequence conservation between NCA-1 and NCA-2 occurs in the four transmembrane domains and in the domain ITf-IV linker. Of the four 122 Table 4. Amino Acid Percent Identity/Similarity Between NCA -1, NCA-2 and rat-NCA Region NCA- l /NCA -2 NCA- l / ra t -NCA NCA-2 / rat-NCA Isoform * N-Terminus 28/40 20/33 18/27 NCA-2 14/22 10/14 A5' NCA-2 Domain I 61/75 43/60 43/60 I-II Linker 45/59 37/44 35/43 Domain II 64/74 52/65 52/66 II-III linker 28/44 39/51 29/44 NCA-1 43/56 28/44 NCA-1 AS Domain UI 75/88 57/71 55/71 III-IV Linker 88/90 63/78 63/78 Domain IV 68/79 48/66 51/65 C-Terminus 42/51 37/47 41/51 rat-NCA A 37/46 41/50 rat-NCA B All 4 Domains 67/79 50/65 50/66 Whole Protein # 57/68 43/57 43/57 * = If alternative splicing occurred in the region examined, both isoforms were used # = The longest open reading frames were used 123 transmembrane domains, domain I was the least conserved (61% identity) and domain III was the most conserved (75% identity) and the highest degree of sequence conservation between NCA-1 and NCA-2 occurs in the domain UI-IV linker (88% identity). In general, the remaining predicted cytoplasmic regions were more variable in their sequence with the amino terminal region being the least conserved (14-20% identity) and the domain I-LT linker being the most conserved (45% identity). Alignment of the longest open reading frames of NCA-1 and NCA-2 (Figure 17) revealed that NCA-1 and NCA-2 are 57% identical and 68% similar on a structural basis to one another. NCA-l/rat-NCA To compare the deduced protein sequences of NCA-1 and rat-NCA, all of the different regions of the channels were aligned separately using the CLUSTAL W algorithm (Higgins and Sharp, 1988) and the percent identities and similarities were determined (Table 4). The similarity analysis showed that the highest degree of sequence conservation between NCA-1 and rat-NCA occurs in the four transmembrane domains and in the domain III-IV linker. Of the four transmembrane domains, domain I was the least conserved (43% identity) and domain m was the most conserved (57% identity) and the highest degree of sequence conservation between NCA-1 and rat-NCA occurs in the domain El-TV linker (63% identity). In general, the remaining predicted cytoplasmic regions were more variable in their sequence with the amino terminal region being the least conserved (28% identity) and the carboxyl terminal region being the most conserved (37% identity). Alignment of the longest open reading frames of NCA-1 and rat-NCA (Figure 17) revealed that NCA-1 and rat-NCA are 43% identical and 57% similar on a structural basis to one another. 124 Figure 17. Alignment Of The NCA-1, NCA-2 and rat-NCA Amino Acid Sequences The predicted amino acid sequence of NCA-1 was aligned with that of NCA-2 and rat-NCA using the CLUSTAL W algorithm. The alignment was then imported in to the Boxshade Program. Regions of identity are shaded in black and regions of similarity are shaded in grey. Transmembrane segments are indicated with lines above the sequence and the additional amino acids present in NCA-1 AS, A5' NCA-2 and rat-NCA B are indicated in red. 125 NCA-1 NCA-2 Rat-NCA MTSTTAKLLGLSACRAAALSTT S H E A P G S A A I I M T K T E R N R F - • V E A Q P V T NCA-1 NCA-2 Rat-NCA NCA-1 NCA-2 Rat-NCA 2 9 5 1 2 8 I S I SSQHKKPiRS|--mESD I E P T S I H N K B T E L S H E KPI I S 2 I S 3 N C A - 1 N C A - 2 Rat-NCA 1 1 5 I S 4 I S 5 N C A - 1 N C A - 2 Rat-NCA 1 5 9 NCA-1 NCA-2 Rat-NCA NCA-1 2 7 5 NCA-2 2 9 9 Rat-NCA 2 5 9 N — . RMK—POEl P | E D L G L S S < I P - L O O P I S 6 NCA-1 NCA-2 Rat-NCA NCA-1 NCA-2 Rat-NCA NCA-1 NCA-2 Rat-NCA I I S I | E | P K - Q S N N S L PACI I I S 2 126 II S3 II S4 NCA-1 NCA-2 Rat-NCA NCA-1 NCA-2 Rat-NCA 473 498 457 NCA-1 523 NCA-2 548 Rat-NCA 506 573 598 556 II S5 II S6 II P-LOOP NCA-1 NCA-2 Rat-NCA NCA-1 NCA-2 Rat-NCA 673 697 656 |HSL| FVETl - IRQQ SDFEFPQRgKSApgR EHGFKV^PJSGRIPSI CSFRl|pSTSS|SCDNPKSPTi NCA-1 NCA-2 Rat-NCA L Y P l KHGgSC _ FEf lQPTKEJgs l ] NCA-1 NCA-2 Rat-NCA NCA-1 NCA-2 Rat-NCA III SI NCA-1 NCA-2 Rat-NCA NCA-1 NCA-2 Rat-NCA III S2 127 I l l S3 I I I S4 N C A - 1 968 N C A - 2 990 R a t - N C A 94 9 N C A - 1 1018 N C A - 2 1 0 4 0 R a t - N C A 999 I I I S5 N C A - 1 1168 N C A - 2 1 1 9 0 R a t - N C A 114 9 jfND N C A - 1 1218 N C A - 2 1 2 4 0 R a t - N C A 1198 I V S I N C A - 1 1268 N C A - 2 1 2 9 0 R a t - N C A 1 2 4 3 Ti I V S2 I V S3 N C A - 1 1318 F f A M s : N C A - 2 1 3 4 0 LFQHA R a t - N C A 12 93 A J J L N — SSKIDVDVQ" Y F A G G — I T ] I V S4 I V S 5 N C A - 1 1368 N C A - 2 1388 R a t - N C A 1 3 2 9 I V P - L O O P N C A - 1 1418 N C A - 2 1438 R a t - N C A 1 3 7 9 ?GEDWNDIMHDCMR 'GEPWNI: >•;•::: 128 I V S6 NCA-1 14 68 NCA-2 1488 R a t - N C A 1428 NCA-1 NCA-2 R a t - N C A NCA-1 1568 NCA-2 1588 R a t - N C A 1528 NCA-1 1618 G NCA-2 1638 N R a t - N C A 1578 K FJGGHPVIHSSGH|SISHEEJ E|QQQEI NCA-1 1668 R GD^|E^A E SB P P P ^J^ A ^^SP I ^N|^ |VAJ Rat -NCA 1618 QPSEDTNAN38JHN|FQPHSBS||QQLLpTiiSDJjpfcRGjAADTGKPQRKI NCA-1 1715 J R K F f V G S S S E H c j f l s R S PEBVQLL|KBNSBRBS|QBNFHlffiNV NCA-2 1711 Q 1 Q -BT^IPRRITLS—QjflCjGS R a t - N C A 1668 @ Q W R | P S A A I A E | ^ V P V N K M S | Y S Q D | E | E E J B | F L A | | P I S r i S f f l s | NCA-1 NCA-2 R a t - N C A 1765 •1P^EJRGM|PFSPML|DKNGEHSPLVITPSLPVPPTH|SPSPLJ 17 37 ^ . teLgEvJED^EDGElSRS|Ff f l s IVDMIoJ 1718 V ^ ^ F G G R T T M K J W C K I ^ P M P J T A S C G SEVJgJKWW NCA-1 1815 NCA-2 1769 R a t - N C A 1753 129 NCA-2/rat-NCA To compare the deduced protein sequences of NCA-2 and rat-NCA, all of the different regions of the channels were aligned separately using the CLUSTAL W algorithm (Higgins and Sharp, 1988) and the percent identities and similarities were determined (Table 4). The similarity analysis showed that the highest degree of sequence conservation between NCA-2 and rat-NCA occurs in the four transmembrane domains and in the domain HI-IV linker. Of the four transmembrane domains, domain I was the least conserved (43% identity) and domain III was the most conserved (57% identity) and the highest degree of sequence conservation between NCA-2 and rat-NCA occurs in the domain IE-TV linker (63% identity). In general, the remaining predicted cytoplasmic regions were more variable in their sequence with the amino terminal region being the least conserved (10-18% identity) and the carboxyl terminal region being the most conserved (41% identity). Alignment of the longest open reading frames of NCA-2 and rat-NCA (Figure 17) revealed that NCA-2 and rat-NCA are 43% identical and 57% similar on a structural basis to one another. Based upon the similarity analysis of the rat-NCA protein with both NCA-1 and NCA-2, no definitive conclusions could be made as to whether rat-NCA is more related to either NCA-1 or NCA-2. Comparison of NCA-1, NCA-2 and rat-NCA with Other VGIC a(i) Subunits To compare the deduced protein sequences of NCA-1, NCA-2 and rat-NCA with representative Ca 2 + and Na + channel a(i) subunits, the putative membrane-spanning domains of the channels were aligned using the CLUSTAL W algorithm (Higgins and Sharp, 1988) and the percent identities and similarities were determined (Table 5). The similarity analysis showed that the voltage-gated Ca 2 + channel family is more closely related to the Na + channel family 130 Table 5. Amino Acid Percent Identity/Similarity Between Four Domain-Type VGIC a ( i) Subunits Channel Region oi(i) subunit NCA-1 NCA-2 rat-NCA Non L-Type UNC-2 20/37 20/39 23/42 CtlA 20/36 22/39 23/40 am 21/37 21/39 23/40 aiR 22/39 23/39 23/41 L-Type EGL-19 23/39 22/40 24/42 a , c 24/40 24/41 24/43 am 22/40 23/41 23/42 a i F 23/40 22/41 24/43 ais 21/40 23/41 23/41 T-Type CCA-1 20/36 21/36 23/38 aia 19/36 20/37 21/38 OtlH 19/36 21/36 21/38 a u 19/37 20/36 21/39 Na Na vl . l 17/34 17/34 18/35 Nav1.2 17/34 18/34 19/37 Nav1.3 18/35 18/34 19/38 NCA NCA-1 100 67/79 50/65 NCA-2 67/79 100 50/66 rat-NCA 50/65 50/66 100 131 Figure 18. Identity Tree Of Four Domain-Type VGIC a(i) Subunits The predicted amino acid sequences of the NCA family were compared pairwise to the amino acid sequences of the different classes of Ca 2 + and Na + channel ct(i) subunits using the CLUSTAL W algorithm and the percent identities were plotted. Only the amino acid sequences of the four conserved domains were used. GenBank Accession Numbers for Ca 2 + channels: rat (XIA , M64373; rat a ] B , M92905; rat ai C , M67515; rat a ! D AF370009; rat a ) E , L15453; human dip, AJ224874; rat ctio, AF290212; rat a m , AF290213; rat an, AF290214; rabbit ais, M23919; UNC-2, AY264781; EGL-19, AF023602; and CCA-1, AY313898. GenBank Accession Numbers for Na + channels: rat Na v l . l , X03638; rat Nav1.2, X03639; rat Nav1.3, Y00766. rat-NCA, AY555273; NCA-1, AY555271 and NCA-2, AY555272. The five four domain-type VGIC ai subunits s present in C. elegans are indicated in red. 132 l N C - 2 N, P/Q-type Ot lA P/Q-type a i B N-typc a i E Novel E G L - 1 9 ais a i c - L-type a i D a i p ( X V I a i G • T-type a i H a n Na v 1.1 N a v 1 . 2 • Na Channel N a v 1 . 3 rat-NCA N C A - 1 N C A - 2 t Novel I 1 1 1 1 20 40 60 80 100 Percent Identity 133 (30% identical), than either are to the NCA family (20% identical) and that on the basis of overall predicted structural conservation and percent similarity and identity, NCA-1, NCA-2 and rat-NCA are likely members of a novel family of four domain-type VGICs (Figure 18). S4 Regions The membrane-spanning S4 regions of Ca 2 + and Na + channel oi(i) subunits contain multiple positively charged residues that are usually separated from each other by two to three hydrophobic residues (Noda et al., 1984; Tanabe et al., 1987; Ellis et al., 1988). The positive charges in the S4 regions are predicted to lie within the membrane and to "sense" changes in membrane potential. Thus, the S4 regions are believed to function as the voltage-sensing mechanism of four domain-type VGICs (Armstrong, 1981; Catterall, 1986; Kontis et al., 1997). Based upon this notion, it has been hypothesized that the positive charges within the S4 regions, as well as other residues, should determine the voltage-dependent properties of four domain-type VGICs (Catterall, 1986; Guy and Seetharamulu, 1986; Kontis et al., 1997; Catterall, 2000a). This hypothesis has been confirmed by in vitro site-directed mutagenesis experiments on Na + channels where charge-neutralizing or charge-conserving substitutions of the positively charged residues in all four S4 domains resulted in changes to the voltage dependence of activation (Stuhmer et al., 1989; Kontis et al., 1997) and inactivation (Stuhmer et al., 1989; Kontis and Goldin, 1997). These studies also found that the positively charged residues in all four S4 regions contribute unequally to the voltage-dependent properties of the channel. While the conserved four domain structures of NCA-1, NCA-2 and rat-NCA are similar to that of Ca 2 + and Na + channel ct(i) subunits examination of their S4 regions shows that the putative voltage sensors in the NCA channels are different from that of both Ca 2 + and Na + channels (Figure 19). In general, the S4 regions of the NCA channels contain fewer positively charged residues than either Ca 2 + or Na + channels. More specifically, in Ca 2 + and Na + channels 134 Figure 19. Comparison of S4 Regions Between the NCA, C a 2 + and Na + Channels The amino acid sequences of the four S4 regions of the NCA family were aligned with the consensus S4 regions of the different classes of Ca 2 + channels (Non L-type, L-type and T-type) and representative Na + Channels (Na*). The positively charged amino acids (lysine (K), arginine (R) and histidine (H)) that constitute the putative voltage sensor are indicated in red and dashes represent amino acid degeneracy. rat-NCA (AY555273), NCA-1 (AY555271) and NCA-2 (AY555272). Non L-type is the consensus sequence for rat a i A (M64373), rat am (M92905) and rat am (L15453). L-type is the consensus sequence for rat am (M67515), rat am (AF370009), human (XIF (AJ224874) and rabbit ais (M23919). T-type is the consensus sequence for rat am (AF290212), rat a m (AF290213) and rat an (AF290214). Na + is the consensus sequence for rat Na vl . l (X03638), ratNav1.2 (X03639) and ratNav1.3 (Y00766). 135 I S4 Non L - t y p e L R T L R A V R V L R P L K L V S G I P S L Q -I I S4 L R A L R L L R I F K - T K Y W - S L R N L W L - t y p e V K A L R A F R V L R P L R L V S G V P S L - -T - t y p e - S A - R T V R V L R P L R A I N R V P S - R I K N C A - 1 I R S I R P F I I I R L I P L W K F K L P K N N C A - 2 L R S A R P F I F L R F I R S I V R F K L P K N R a t - N C A L R I P R P L I M I R A F R I Y F R F E L P R T - R C - R L L R - F K - T R - W - S L S N L V A K - S V L R T - R L - R V L K L V R F - P A L R R Q F Q T F R L L R L I K A S P I L E D F V W K I F F Q V F R I A R L I K A S P M L E D F V Y K I F F Q V L R W R L I K I S P A L E D F V Y K I F N a + L R T F R V L R A L K T I S V I P G L K T I V G L R S F R L L R V F K L A K S W P T L N M L I K I I I S4 N o n L - t y p e I K S L R V L R V L R P L K T I K R L P K L K A L - t y p e V K I L R V L R V L R P L R A I N R A K G L K H T - t y p e L R V - R L L R T L R P L R V I S R A - G L K L I V S4 - S F L R L F R A A R L I K L - R Q G Y T I R I K - - F F R L F R V M R L - K L L S R - E G - R T K I R I M R V L R I A R V L K L L K M A - G M R A N C A - 1 A Q F L M I C R A M R P L R I Y T L V P H I R R N C A - 2 A Q L L M V C R A M R P L R V Y A L I P H I R R R a t - N C A A Q L L M V L R C L R P L R I F K L V P Q M R K G Y L W I L R F F T I A S R N S T L K M L M L G Y L W I L R F F T I A G R K S T L K M L M L G A C V I V F R F F S I C G K H V T L K M L L L N a + I K S L R T L R A L R P L R A L S R F E G M R V F R V I R L A R I G R I L R L I K F A K F I R T 136 the domain IIS4 regions contain five (L-Type), six (Non L-Type, some T-types and Na*) or seven (some T-Type) positively charged residues, whereas the same S4 regions in the NCA channels contain only four positive residues. Additionally, in Ca 2 + and Na + channels the domain IV S4 regions contain six (Non L-Type and L-Type), seven (T-type) or eight (Na*) positively charged residues, whereas the same S4 regions in the NCA channels contain either three (NCA-1) or four (NCA-2 and rat-NCA) positive residues. Furthermore, the distribution of the positively charged amino acids within the S4 regions of the NCA channels does not follow the pattern of every third or fourth position seen in the S4 regions of most Ca 2 + and Na+ channels. Based upon the aforementioned in vitro site-directed mutagenesis experiments on Na + channels, it is hypothesized that NCA-1, NCA-2 and rat-NCA are members of a novel family of VGICs that may exhibit unique activation and inactivation properties (see Discussion). P-Loop Regions The involvement of the P-Loop region of four domain-type VGICs in ion selectivity was first demonstrated by in vitro site-directed mutagenesis experiments on Na + channels, where it was shown that making the double amino acid substitution K1422E and A1714E in domain HI and domain IV P-Loops, respectively changed the ion selectivity of the Na + channel (DEKA to DEEE) from monovalent to divalent selective (Heinemann et al., 1992). Furthermore, making the amino acid substitution of just A1714E in the domain IV P-Loop changed the ability of the Na + channel (DEKA to DEKE) to properly discriminate between monovalent cations (Heinemann et al, 1992). These results on the Na + channel implied that the corresponding amino acid residues in Ca channels (EEEE or EEDD) may also be important in determining ion selectivity. This was confirmed by in vitro site-directed mutagenesis experiments which showed that the glutamate residues (E) in the P-Loops of each of the four domains are critical in determining the ion selectivity of L-type HVA Ca 2 + channels (Kim et al., 1993; Tang et al., 1993; Yang et al., 137 1993). Mutations of the glutamate (E) residues dramatically decreased the selective permeation of divalent over monovalent cations (Kim et al., 1993; Yang et al., 1993; Yatani et al., 1994; Ellinor et al., 1995; Parent and Gopalakrishnan, 1995). It appears that the high affinity binding site(s) responsible for Ca 2 + selectivity is formed by these four glutamate residues in HVA Ca 2 + channels or the two glutamate and two aspartate residues in LVA Ca 2 + channels and that each of these negatively charged residues contributes differently to Ca 2 + affinity, selectivity and permeation. For example, mutations to the domain m glutamate have the greatest effect on ion selectivity and permeation, as making the amino acid substitution E1086K at this position in the pore of the cardiac Ca 2 + channel (EEEE to EEKE) changed the ion selectivity from divalent to monovalent selective (Tang et al., 1993; Yang et al., 1993; Ellinor et al., 1995). While the conserved four domain structure of NCA-1, NCA-2 and rat-NCA are similar to that of Ca 2 + and Na + channel oi(i) subunits, examination of their pore regions shows that the ion selectivity motif in the NCA channels (EEKE) is different from that of both Ca 2 + (EEEE or EEDD) and Na + channels (DEKA). Figure 20 shows that unlike Ca 2 + channels but similar to Na + channels, the NCA-1, NCA-2 and rat-NCA channels contain a lysine (K) in the P-Loop of domain HI. Furthermore, like HVA Ca 2 + channels, but unlike Na + channels, NCA-1, NCA-2 and rat-NCA contain a glutamate (E) in the P-Loop of domain IV. Based upon previous in vitro site-directed mutagenesis experiments, it is hypothesized that NCA-1, NCA-2 and rat-NCA are members of a novel family of VGICs that may exhibit unique ion selectivity properties (see Discussion). rat-nca Regional RNA Expression To examine the expression pattern of rat-nca, total RNA was isolated from various tissues of adult male and female rats. RT-PCR of total RNA was performed using primers specific to the carboxyl tail region of rat-NCA. The primer KH83 was used for first strand cDNA 138 Figure 20. P-Loop Comparison Between the N C A , C a 2 + and N a + Channels The amino acid sequences of the four P-Loop regions of the NCA family were aligned with the consensus P-Loop sequences of the different classes of Ca 2 + channels (Non L-type, L-type and T-type) and representative Na + Channels (Na*). The amino acids that constitute the ion selectivity filter are indicated in bold and dashes represent amino acid degeneracy. NCA-1 (AY555271), NCA-2 (AY555272) and rat-NCA (AY555273). Non L-type is the consensus sequence for rat a i A (M64373), rat am (M92905) and rat am (L15453). L-type is the consensus sequence for rat am (M67515), rat am (AF370009), human ociF (AJ224874) and rabbit a i S (M23919). T-type is the consensus sequence for rat a ] G (AF290212), rat a m (AF290213) and rat an (AF290214). Na + is the consensus sequence for rat Na vl. 1 (X03638), rat Nav1.2 (X03639) andratNav1.3(Y00766). 139 I P - L O O P N o n L - t y p e F D N I - F A — T V F Q C I T M E GWT Y L - t y p e F D N F - F - M L T V - Q C - T M E GWTDVLY T - t y p e F D N I G Y A W I - I F Q V I T L E GWV- IMY I I P - L O O P F D T F P A A I - T V F Q I L T G E DWN-VMY F D - F P Q - L — V F Q - L T G E DWN-VMY F D S L L W A I V T V F Q I L T Q E DWN-VLY N C A - 1 F N D F G A S V F T V Y L A A S E E GWVYVLY N C A - 2 F S D F A S S L F T V Y L A A S Q E GWVYVLY R a t - N C A F N E I G T S I F T V Y E A S S Q E GWVFLMY F R T F P Q A F M S M F Q I I T Q E G W T D F W F T N F A V A F M S M F Q I I T Q E G W T D W I F T T F P R A F M S M F Q I L T Q E GWVDVMD N a + F D T F S W A F L S L F R L M T Q D - W E N L Y Q M — F F H S F L I V F R V L C G E WIETMWD I I I P - L O O P N o n L - t y p e Y D N — W A L L T L F T V S T G E G W P - V L -L - t y p e F D N V L - A M M - L F T V S T F E G W P - L L Y T - t y p e F D N L G Q A L M S L F V L - S K D GWV- IMY I V P - L O O P F R - F L M L L F R S A T G E A W — I ML F Q T F P Q A V L L L F R C A T G E A W Q - I - L F - N A F L T L F - V S T G D NWNGIMK N C A - 1 F D H V G N A M L A L F E T L S F K GWNVIRD N C A - 2 F D H I G N A M L A L F E T L S Y K GWNWRD R a t - N C A F D N V G N A M L A L F E V L S L K GWVEVRD F R T A S E A L W L F R C L T G E DWNDIMH F R N G R E A L W L F R S V T G E DWNDIMH F S S A G K A I T V L F R I V T G E DWNKIMH N a + F D N V G - G Y L - L L Q V A T F K GWMDIMY F E T F G N S M I C L F Q I T T S A GWDGLLA 140 synthesis and the upstream KH61-KH84 primer set was used for PCR amplification. The primer set KH61-KH84 (bp 4785-5208 of rat-nca A cDNA) was used for rat-nca expression pattern analysis because they span over 5 exons and -7.3 kb of genomic sequence. As a positive RNA control, RT-PCR was also performed for rat a-tubulin using the primer JM92 for first strand cDNA synthesis and the JM91-JM92 primer set for PCR amplification and the 223 bp product was found in all samples. The RT-PCR products were separated on a 2% agarose gel, subjected to Southern blotting and probed with the internal primer KH62 (rat-nca) or JM93 (a-tubulin) that had been y-32P labeled to confirm the identity of the respective RT-PCR products. All positive RT-PCR products were subcloned and sequenced to confirm their identity. RT-PCR in combination with Southern blot analysis revealed that rat-nca is expressed in all brain regions (pons/medulla, cerebellum, striatum, hypothalamus/thalamus, cortex and olfactory bulb) and organs examined (seminal vesicles, ovaries, eye, kidney, adrenal gland, heart, spleen and lung) (Figure 21). Furthermore, two different splice variants of rat-nca were detected. A smaller rat-«ca A isoform (424 bp RT-PCR fragment) was expressed in all tissues examined, whereas a larger rat-nca B isoform (520 bp RT-PCR fragment) was expressed in all tissues examined except for kidney, adrenal gland and heart. By comparing the sequences of the rat-nca RT-PCR products with the predicted rat-nca genomic sequence in GenBank, it was determined that the rat-nca B isoform is due to the inclusion of an extra 96 bp exon in the carboxyl tail region (see Figure 10). The observed expression pattern of rat-nca in this study is different than that previously reported (Lee et al., 1999a) and may suggest a more widespread role for the NCA channel in mammals. Using Northern blot analysis, another group observed Rb21 (rat-nca) expression only as a single 6.9 kb fragment. The inability to detect different sized transcripts that are the result of small insertions or deletions like the 96 bp insertion in my rat-nca B isoform exemplifies the usefulness of using RT-PCR for examining gene expression. In addition, Rb21 was found to be 141 Figure 21. RNA Expression Pattern of rat-nca as Determined by RT-PCR Southern blot of RT-PCR products generated using primers designed against rat-nca carboxyl tail (KH61-KH84 bp 4785-5208) and rat a-tubulin (JM91-JM92) for various tissues from adult male and female rats. The Southern blot was probed with a y-32P labeled internal primer KH62 (rat-nca) or JM93 (a-tubulin). The 223 bp a-tubulin product was found in all samples tested. Two different RT-PCR products, rat-nca A isoform (424 bp) and rat-nca B isoform (520 bp) were generated. All positive RT-PCR products were subcloned and sequenced to confirm their identity. 142 143 expressed in rat brain, heart and human pancreas and not in lung, skeletal muscle, testis, kidney, liver or placental tissue (Lee et al., 1999a). This discrepancy between the expression patterns observed for the two studies may reflect the different sensitivities of the two experimental approaches used. While not all of the same tissues were examined in each of these two studies, BLAST searches of the EST database have identified two related nca clones from mouse eye (CF733139) and human lung (BE220480) supporting the observed expression of rat-nca in these tissues. Electrophysiological Analysis of HEKtsA201 Cells Tranfected with Rat-nca A Functional analysis of the rat-NCA A channel was performed by heterologous expression in the HEK tsA201 cell line. The rat-nca A cDNA was cotransfected alone or with either the Ca 2 + channel accessory subunits a28-l and Pu, (or p2a) or with the Na + channel Pi accessory subunit. Functional expression of the rat-NCA A channel was evaluated using different experimental protocols and different recording solutions with various compositions. In the first experimental protocol, the external recording solution contained (in mM): NaCI (116), KC1 (5), CaCl2 (1.8), MgCl 2 (1), HEPES (10) and glucose (5.5) and the internal recording solution contained (in mM): Kaspartate (105), EGTA (5), CaCl2 (5), MgCl 2 (1) and HEPES (10). In this experiment, the standard K + channel blockers TEAC1 and CsCl were not included in the external and internal recording solutions, respectively in order to address the possibility that the rat-NCA A channel may permeate K + ions and may be blocked by the addition of either TEAC1 and/or CsCl to the solutions. In addition, high levels of CaCl2 were added to the internal recording solution in order to address the possibility that elevated levels of intracellular Ca 2 + may be required to activate the rat-NCA A channel (Colquhoun et al, 1981). Lastly, Kaspartate was used in place of KC1 in the internal recording solution in order to eliminate endogenous CI" currents in HEKtsA201 cells. 144 When the membrane potential was stepped from a holding potential of -90 mV to various test potentials ranging from -60 to +90 mV for 20 ms and then repolarized to -30 mV for approximately 50 ms, the main component of the whole cell current was an outward current whose characteristics were similar to delayed rectifier K + currents observed in mock transfected or in non-transfected HEK cells and thus could not be attributed to the rat-NCA A channel. In the second experimental protocol, the external recording solution contained (in mM): NaCI (100), TEAC1 (35), HEPES (10), EGTA (2) and glucose (10) and the internal recording solution contained (in mM): CsCl (105), TEAC1 (25), CaCl2 (1), EGTA (11) and HEPES (10) and a phosphorylation cocktail consisting of 5 mM Mg-ATP, mycrocystin and phosphocreatin. In this experiment, CaCl2 was left out of the external recording solution in order to address the possibility that the rat-NCA A channel may be activated by low levels of extracellular Ca 2 + as has been described for certain types of Ca2+-activated non-selective cation channels (Chu et al., 2003). Mg-ATP, mycrocystin and phosphocreatin were added to the internal recording solution to prevent potential run-down or wash out of measured currents and to address the possibility of second messenger pathways involving kinase activity that may be necessary to activate and/or maintain the rat-NCA A channel activity. Whole cell currents were measured using two different voltage protocols. The first method of measuring whole cell current was with voltage ramps from -100 mV to +100 mV. The second method consisted of 200 ms steps from -60 mV to +30 mV from a holding membrane potential of-120 mV. In both scenarios, a slow transient inward current was activated at -60 mV and the peak current value was obtained at -40 mV. However, this current was observed also in mock transfected cells and thus could not be attributed to the rat-NCA A channel. 145 The aforementioned protocols were also attempted with the NCA-2 channel alone and under the experimental conditions outlined above, although no currents were obtained that could be directly attributed to the NCA-2 channel. Rat-NCA Expression in Transfected H E K tsA201 Cells and Rat Brain Homogenates In order to examine whether the lack of functional currents obtained from whole cell patch clamp recordings of HEK tsA201 cells transiently transfected with rat-nca A was due to protein expression problems, polyclonal antibodies were generated against a bacterial fusion protein specific to Rat-NCA. The channel region chosen was the intracellular linker between domains I and II and was expressed as a fusion with the RsaA protein from Caulobacter. The corresponding sera, designated BC188 and BC189, specifically recognized the fusion protein in Western blotting experiments (data not shown). However, a band of the expected size was not detected on Western blots of rat brain protein lysates or from protein lysates obtained from HEK cells transiently transfected with the rat-nca A cDNA using these sera (data not shown). Similarly, reproducible cell staining results were not obtained for HEK cells transiently transfected with the rat-nca A cDNA (data not shown). This inability to detect the rat-NCA channel using these different experimental approaches may be due either to a lack of channel expression within the surrogate expression system or the inability of the polyclonal antibodies in the sera to recognize the native rat-NCA channel protein. In vitro translation of the rat-nca A cDNA resulted in the production of a protein product of ~ 200 kDa (data not shown) demonstrating that the production of a full-length rat-NCA A channel was possible from the rat-nca A cDNA. In order to address whether the inability of the sera to recognize the rat-NCA channel was due to the lack of antibodies that could recognize the native channel, a FLAG epitope was introduced into the rat-nca A cDNA as a carboxy terminal fusion. By transiently expressing this 146 channel fusion construct in HEK cells and staining with the M2 anti-FLAG antibody the issue of whether a full-length rat-NCA channel was being expressed in this surrogate system could be addressed. However, the rat-NCA A::FLAG channel protein was not detected in either Western blots of protein lysates obtained from transiently transfected HEK cells, nor was it detected by cell staining (data not shown). Although these results were not positive, they suggest the possibility that a full-length rat-NCA A channel is not being synthesized and/or properly processed in HEK cells. The presence of the highly negatively charged FLAG epitope (DYKDDDDK) at the end of the channel may affect its expression as has been observed when the FLAG epitope has been added to a number of different Ca 2 + channel ai subunits (E. Bourinet, pers. comm.). This issue could be further addressed by generating additional channel fusions using other epitopes, such as GFP, as well as by generating antibodies directed against different regions of the rat-NCA A channel. 147 Chapter 4. Genetic and Phenotypic Analysis of nca-1 and nca-2 Mutant Strains Background C. elegans is amendable to genetic manipulation and can be used to generate physiologically relevant mutations in four domain-type VGICs and their interacting proteins. The phenotypes of mutant animals can provide clues to the physiological function of the gene product of interest. For example, reduction-of-function and loss-of-function mutations provide insight into the normal role of the ion channel proteins through analysis of the biological processes that are perturbed. Furthermore, missense mutations which result in the expression of an altered ion channel with changes to its biochemical, electrophysiological and/or pharmacological properties will aid in the identification of amino acids that are critical for these functions. Once mutant strains have been established, they can be subjected to further genetic and molecular analysis. Genetic screens for secondary mutations that either "suppress" or "enhance" the phenotype of the original mutation can be used to dissect stracture-function relationships within the gene of interest, as well as to identify interacting genes. Furthermore, the search for possible genetic and functional interactions between four domain-type VGICs and other gene products can be aided by the introduction of other gene mutations into the background of animals that already have mutations in VGIC genes. The nervous system of the C. elegans adult hermaphrodite is relatively simple and is composed of only 302 neurons and 56 support cells, yet it generates and regulates a repertoire of 148 simple behaviors including, locomotion, mechanosensation, touch avoidance, chemo and thermotaxis, egg-laying, mating, pharyngeal pumping and defecation (Chalfie and White, 1988; Avery and Thomas, 1997; Bargmann and Mori, 1997; Driscoll and Kaplan, 1997). A number of behavioral paradigms have been developed to assess changes to these behavioral characteristics in both wild-type and mutant animals (Trent et al., 1983; Chalfie and White, 1988; Thomas, 1990) and have proven to be powerful experimental systems for the dissection of the underlying neural signaling pathways and the functions of specific genes within these neural circuits. Therefore, a combined genetic and behavioral approach will help to define the physiological functions of nca-1 and nca-2 and help to identify interacting proteins. In the following two sections I describe the locomotory and defecation motor programs in C. elegans which have proven to be useful tools in gaining knowledge about the biological roles and physiological functions of VGICs in C. elegans and were used as behavioral paradigms to explore the roles of the new four domain-type VGIC ai subunits identified in this study. The Locomotion Circuit in C. elegans Through the use of genetic, molecular and cell ablation techniques, the neural circuits controlling locomotion in C. elegans have been determined. Locomotion in C. elegans is accomplished by the coordinated contraction of the dorsal and ventral body wall muscles which are innervated by different classes of excitatory cholinergic (classes A, B and AS) (Alfonso et al., 1993; Driscoll and Kaplan, 1997) and inhibitory GABAergic motor neurons (class D) (Mclntire et al., 1993b; Driscoll and Kaplan, 1997; Bamber et al., 1999) whose cell bodies are located in the ventral nerve cord. Each class of motor neurons are further subdivided based upon whether they innervate dorsal (AS, DA, DB and DD) or ventral (VA, VB and VD) body wall muscles (White et al., 1976,1986). In order to generate sinusoidal movement, contraction of dorsal body wall muscles must be accompanied by the simultaneous relaxation of ventral body 149 wall muscles and vise versa. For example, to bend the body in the ventral direction, ventral body wall muscles contract in response to receiving excitatory cholinergic neurotransmission from either VA or VB motor neurons, whereas dorsal body wall muscles relax in response to receiving inhibitory GABAergic neurotransmission from DD motor neurons. Furthermore, the class A and B motor neurons form two synaptic connections, one with the body wall muscle and the other with a dendrite from a class D motor neuron that innervates body wall muscle on the opposite side of the worm (White et al., 1986). Thus, movement in C. elegans is achieved by an altering pattern of dorsal and ventral contraction and a corresponding cross-talk between excitatory and inhibitory motor neurons (White et al., 1986) (Figure 22A). In the laboratory environment, wild-type animals on agar plates exhibit a characteristic pattern of locomotion that consists mainly of forward movement that is occasionally interrupted by brief periods of reversals or backwards movement. Furthermore, the worm can also be specifically instructed to change its direction of motion through activation of specialized sensory neurons (Driscoll and Kaplan, 1997). For example, C. elegans has specialized receptors that sense touch, vibration, temperature, different types of chemicals and the presence of food and can change its direction of movement in response to these environmental factors (Chalfie et al., 1985; Bargmann et al., 1990; Wicks and Rankin, 1995; Driscoll and Kaplan, 1997). Through the use of cell-ablation experiments, it has been determined that there are two different but interconnected neuronal circuits that control forward and backward locomotion and the "command center" for these two circuits together consist of only five pairs of neurons, called the command interneurons (Chalfie et al, 1985). The forward locomotion circuit consists of the AVB and PVC command interneurons and the class B motor neurons, whereas the backward locomotion circuit consists of the AVA, AVD and AVE command interneurons and the class A motor neurons (Chalfie et al., 1985) (Figure 22A). In the simplest model of locomotion, the command neurons that control forward and backward movement can be modeled as just two 150 Figure 22: The Locomotory Circuit in C. elegans A) Locomotion in C. elegans is accomplished by the coordinated contraction of the dorsal and ventral body wall muscles which are innervated by different classes of motor neurons (inverted triangles) which are subdivided based upon whether they innervate dorsal (DA, DB and DD) or ventral (VA, VB and VD) body wall muscles. The circuit consisting of the AVB and PVC command interneurons (rectangles) and the class B (DB and VB) cholinergic motor neurons (white inverted triangles) drives forward locomotion, whereas the circuit consisting of the AVA, AVD and AVE command interneurons (rectangles) and the class A (DA and VA) cholinergic motor neurons (white inverted triangles) drives backwards movement. Coordinated movement is obtained by the simultaneous inhibition of the contralateral body wall muscles by the appropriate class D (DD and VD) GABAergic motor neurons (grey inverted triangles). (Figure adapted from Driscoll and Kaplan, 1997). B) A simplified model of the locomotory circuit where the command neurons that control forward (grey) and backward (white) movement can be modeled as just two neurons that have inhibitory connections with one another (red) and excitatory connections with their respective class of motor neurons (black arrows). The activity of these two neural circuits can be altered through inputs from the sensory neurons (arrows on the neurons) and can be made to oscillate between the forward and backward states by the integration of the excitatory and inhibitory inputs received. (Figure taken from Zheng et al, 1999) 151 A 152 neurons (one for each of the two circuits) that have inhibitory connections with one another and excitatory connections with their respective class of motor neurons (Chalfie and White, 1988; Zheng et al., 1999). In addition, the activity of these two neural circuits can be altered through inputs from the sensory neurons by way of primary and secondary interneurons, the presumed sites of sensory integration (Figure 22B). Consequently, this simplified two neuron circuit can be made to oscillate between the forward and backward states and the time spent in any one state is determined by the sum of all the excitatory inputs received by a given circuit, as well as the strength of the inhibitory input received from the opposite circuit (Zheng et al, 1999). Thus, mutations to genes (such as VGICs) that affect the properties of the neurons within these two circuits or any of their upstream or downstream components may result in changes to the wild-type pattern of locomotion (Hobert, 2003). The Defecation Motor Program in C. elegans Another well characterized behavior in C. elegans is the defecation motor program which consists of a series of three independent muscle contractions that occur in sequence approximately every 45 to 50 seconds (Thomas, 1990; Liu and Thomas, 1994; Iwasaki et al., 1995). Through the direct observation of wild-type and mutant animals, it has been determined that the defecation motor program begins with a posterior body wall contraction (pBoc) which causes an increase in internal pressure at the site of muscle contraction and forces the intestinal contents anteriorly (Thomas, 1990; Avery and Thomas, 1997). Approximately one second from the beginning of the pBoc, the posterior muscles relax and the intestinal contents flow back and collect near the anus. Around this time, the body wall muscles near the head contract in all four quadrants and force the pharynx into the anterior of the intestine (Thomas, 1990; Avery and Thomas, 1997). This second contraction step, called the aBoc (anterior body muscle contraction), is thought to help collect the intestinal contents near the anus. At about the time the aBoc reaches 153 its maximum, the expulsion event occurs through the simultaneous contraction of the enteric muscles. This last event is called the Emc (expulsion muscle contraction) and involves the contraction of the anal depressor, the anal sphincter and the last two intestinal muscles (Thomas, 1990; Avery and Thomas, 1997) (Figure 23). The precision of the defecation motor program demands coordination between both the initiation and execution of the muscle contraction steps of this cycle. In order to achieve this coordination, it is presumed that at least some of these events are under the control of the nervous system (Avery and Thomas, 1997). Laser ablation experiments have shown that the Emc step of the defecation motor program is under the control of the GABAergic motor neurons, AVL and DVB which innervate the enteric muscles (White et al., 1983; Avery and Thomas, 1997). Killing of both AVL and DVB eliminates contraction of the enteric muscles and results in a substantial increase in the failure rate of expulsion (Thomas, 1990; Mclntire et al., 1993a; Mclntire et al., 1993b). Since the maintenance of the defecation motor program requires inputs from both the muscles that contract during any step of the defecation motor program, as well as from inputs from AVL and DVB which regulate the aBoc and Emc steps (Mclntire et al., 1993b; Avery and Thomas, 1997), mutations to genes involved in these biological processes can lead to defects in the defecation motor program. In contrast, to the Emc step that requires neuronal control from AVL and DVB, the timing of the cycle and initiation of the pBoc step does not seem to require neuronal input. Support for the lack of nervous control over the timing of the defecation cycle comes from the observation that mutants with disrupted neurotransmission do not display altered timing and that extensive neuronal ablation experiments have also been unsuccessful at altering the timing of the defecation cycle (Dal Santo et al., 1999). Instead, oscillating Ca 2 + levels in the intestine during the defecation motor program may partially control the timing of the cycle as Ca 2 + levels peak 154 Figure 23. The Muscle Contractions of the Defecation Motor Program in C. elegans The defecation motor program consists of three well defined steps involving the coordinated contraction of different muscle groups. The program begins with a posterior body wall contraction (pBoc) that forces the intestinal contents anteriorly. Approximately one second later these muscles relax and the anterior body wall muscles contract (aBoc) collecting the intestinal contents near the anus. At about the time the aBoc reaches its maximum, the expulsion event occurs by contraction of the enteric muscles (Emc). The defecation motor program is repeated every 45 to 50s. (Figure taken from Strange, 2003). 155 156 Figure 24. A Model Illustrating the Role of Intracellular Ca in Regulating the Timing of the Defecation Motor Program in C. elegans According to this model, oscillating levels of Ca 2 + in the intestine mediated by the HVdependent Ca 2 + release from internal stores causes the release of an unknown messenger (?) that initiates contraction of the posterior body wall muscles and the pBoc event. IP3, inositol 1,4,5-triphsophate; IP3R, inositol 1,4,5-triphsophate receptor; PLC, phospholipase C. (Figure taken from Strange, 2003). 157 158 just prior to the pBoc step and may serve as a signal to start the program through the release of an unknown messenger (Dal Santo et al., 1999). In support of this hypothesis, mutations to the itr-1 gene in C. elegans which encodes an inositol 1,4,5-triphsophate (IP3) receptor that is expressed in intestinal cells change the timing of the defecation cycle presumably by altering the Ca 2 + dynamics in the intestine and, consequently, the ability to initiate the cycle (Dal Santo et al., 1999) (Figure 24). Results ' Isolation of Deletion Mutations in the nca-1 and nca-2 Genes Since the nca-1 and nca-2 genes were first identified by screening the C. elegans genome database, no visible mutations had previously been reported for either gene. In order to explore their biological roles and physiological functions, a recently developed PCR-based gene knockout approach was used to isolate putative null alleles of nca-1 and nca-2 (Jansen et al., 1997; Liu et al., 1999). The two knockout alleles, nca-l(gk9) and nca-2(gk5), were generated by the C. elegans Gene Knockout Consortium at the University of British Columbia. The mutant strains VC12 nca-1 (gk9)IV and VC9 nca-2(gk5) were each backcrossed six times with N2 males in order to reduce the likelihood of any additional mutations elsewhere in their genomes. Backcrossing of VC12 nca-1 (gk9)IV and VC9 nca-2(gk5)III resulted in the generation of the TS61 nca-1 (gk9)IV and TS46 nca-2(gk5)III mutant strains, respectively. Molecular Analysis of the nca-1 (gk9) and nca-2(gkS) Deletion Alleles Molecular analysis of the TS61 nca-1 (gk9)IV knockout strain indicated that the molecular lesion in nca-1(gk9) is a 2287 bp deletion together with a 128 bp insertion of duplicated genomic sequence from elsewhere in the genome at the deletion breakpoint. The deficiency spans from 159 the end of intron 13 (bp 15464 of cosmid CI 1D2 (AF045640)) to the middle of intron 18 (bp 17750) and removes the last half of the domain n-ffl linker to the domain UJ S5 transmembrane segment from the putative NCA-1 ion channel protein (Figure 25A). Based upon the size of the deletion, the region of the channel that is deleted and the fact that the subsequent reading frame is altered as a result of the deficiency and insertion, it is likely that the nca-1 (gk.9) is a null allele. Similarly, molecular analysis of the TS46 nca-2(gk5) knockout strain indicated that the molecular lesion in nca-2(gk5) is a 2970 bp deletion. The deficiency spans from the end of intron 7 (bp 16483 of cosmid C27F2 (U40419)) to the beginning of exon 16 (bp 19452) and removes the domain II P-Loop to the beginning of the domain HI S6 transmembrane segment from the putative NCA-2 channel (Figure 25B). Based upon the size of the deletion, the region of the channel that is deleted and the fact that the subsequent reading frame is altered as a result of the deficiency, it is likely that nca-2(gk5) represents a null allele. Traditionally, genetic analysis in C. elegans has been pursued by forward genetics in which animals are mutagenized and mutant phenotypes (e.g. uncoordinated movement) are selected by visual inspection in subsequent generations. One difficult aspect of forward genetics is to subsequently define the affected gene. On the other hand, reverse genetics starts with a well-defined gene (e.g. nca-1 or nca-2) and the difficult aspect is to define a corresponding mutant phenotype. This can be especially difficult when no visible phenotype is readily apparent. In this study, both the TS61 nca-1 (gk9)IV and TS46 nca-2(gk5) knockout strains were examined for any visible locomotion or morphological abnormalities. For example, neither strain exhibited a noticeable change to their sinusoidal pattern of movement nor their ability to move in the forward or backwards direction. Furthermore, the length and shape of each strains appeared to be normal, as well as the physical integrity of the cuticle. Thus, neither mutant strain exhibited any readily noticeable mutant phenotype and a more thorough phenotypic analysis of the mutant strains was required (see below; Phenotypic Analysis of nca Mutant Strains). 160 Figure 25. Diagrams Illustrating the Molecular Lesions in nca-l(gk9) and nca-2(gk5) For all gene diagrams, exons and introns are represented as boxes and lines, respectively. Shown below the gene structures are the predicted proteins where the transmembrane segments are indicated as boxes and the cytoplasmic and extracellular domains are indicated as lines. A) The molecular lesion in nca-1(gk9) is a 2287 bp deletion (from bp 15464 to bp 17750 of cosmid CI 1D2 (AF045640)) together with a 128 bp insertion at the deletion breakpoint. The deficiency spans from the end of intron 13 to the middle of intron 18 and removes the last half of the n-HI linker to the domain HI S5 transmembrane segment from the putative NCA-1 channel. The region of the gene deleted in nca-1 (gk9) and the corresponding NCA-1 channel are indicated with red boxes. B) The molecular lesion in nca-2(gk5) is a 2970 bp deletion (from bp 16483 to bp 19452 of cosmid C27F2 (U40419)). The deficiency spans from the end of intron 7 to the beginning of exon 16 and removes the domain II P-Loop to the beginning of the domain III S6 transmembrane segment from the putative NCA-2 channel. The region of the gene deleted in nca-2(gk5) and the corresponding NCA-2 channel are indicated with red boxes. 161 162 nca-1 Gene Expression Pattern One way to narrow down the broad spectrum of possible behavioral paradigms that could be used to search for a mutant phenotype is to determine the cellular expression pattern of the gene of interest. While the rigorous identification of specific cell types by following cell lineages through embryonic and postembryonic divisions is a very time consuming process, most of the cells in C. elegans are found in reproducible positions and a combination of morphological cues and position can be used to identify individual cells circumventing the need to follow cell lineages (Bargmann and Avery, 1995). Once cell identity and cell function has been determined, one can use specific behavioral paradigms that exploit defects in those particular cells. To examine the cellular expression pattern of the nca-1 gene, the nca-1 promoter: :GFP reporter construct (pnca-1 :.G¥P) was generated by two rounds of PCR using the primer set KH79OS-KH80OS and N2 genomic DNA as a template for the first round and the nested primer set KH79-KH80. The pnca-1 ::GFP reporter construct contained 3418 bp of putative promoter region upstream of the start site in exon 1 and had GFP fused in-frame with respect to the nca-1 open reading frame at exon 1 (Figure 11A). The pnca-1 :.G¥? reporter construct and the co-injection marker plasmid plin-15(+) were injected into the syncytial gonads of MT8189 lin-75f«765te^hermaphrodites. Transgenic lines were subsequently established and the strain, TS4 vaEx3 was chosen as the source strain for integration of the extrachromosomal array. After integration, the transgenic strain TS48 vals6 was examined for GFP expression. GFP fluorescence was found to be primarily localized within the nervous system (Figure 26). Expression was observed late in embryogenesis and continued throughout development to the adult stage. The pnca-7::GFP reporter construct was expressed in a number of neurons in the pharyngeal nervous system including the II interneurons (I1L/R) the motor/ interneuron MI and the M2 motor neurons (M2L/R). GFP expression was also observed in numerous neurons in the nerve ring of which only a few have been tentatively identified. The p«ca-7::GFP reporter 163 Figure 26. Expression of the nca-1 promoter::GFP Fusion Construct (p«ca-/::GFP) in Transgenic Animals In all panels, the anterior of the animal is positioned to the left and the dorsal side is topmost. All panels represent a merge of DIC and GFP fluorescent images of TS48 vals6 transgenic animals expressing the pnca-l::GFP fusion construct. A) The overall GFP fluorescence pattern exhibited by a typical L3 hermaphrodite (left side view). GFP expression is observed in a subset of neurons in the pharyngeal nervous system including the II interneurons and the motor/interneuron MI (not in focus). Expression of the GFP reporter construct is also observed in numerous unidentified nerve ring interneurons {filled arrowhead), as well as in a subset of motor neuron cell bodies present in the ventral nerve cord (VNC, double arrowhead). Additional GFP expression is observed in the lateral interneuron SDQL (not in focus), as well as in the DVB neuron of the dorso-rectal ganglion and in the ALN and PLN interneurons of the lumbar ganglion. Scale bar = 50 urn. B) An enlarged view of the region encompassed by the red bracket (}) in panel A. The GFP fluorescence pattern exhibited in the ventral nerve cord of a typical L3 hermaphrodite around the hypodermal cells P9 and P10 (left side view). GFP expression is observed in the class A (VA10, DA7 and VA11), class B (VB11 and DB7) and class AS (AS9 and AS 10) cholinergic motor neurons, but not in the class D (DD5, VD10 and VD11) GABAergic motor neurons. GFP expression is faint in cell DA7 and cell VD10 is out of the plane of focus. Scale bar = 1 urn. C) An enlarged view of the region encompassed by the blue bracket (]) in panel A. The GFP fluorescence pattern exhibited in the pre-anal, dorso-rectal and lumbar ganglia of a typical TS48 L3 hermaphrodite (left side view). GFP expression is observed in cholinergic motor neurons of the pre-anal ganglia (arrow), the DVB neuron required for defecation and in the ALN and PLN interneurons (not in focus). Scale bar =10 um. 164 165 Table 6. Summary of Tentatively Identified Neurons Expressing the pnca-1 ::GFP Reporter Construct Cell Location (Cell Type) Function Reference I1L Pharynx Anterior Bulb (Intemeuron) Connected to Extraphyngeal Nervous System via RIPs Modulates Pumping Rate in Absence of Bacteria (Avery and Thomas, 1997) I1R Pharynx Anterior Bulb (Intemeuron) Connected to Extraphyngeal Nervous System via RIPs Modulates Pumping Rate in Absence of Bacteria (Avery and Thomas, 1997) M2L Pharynx Posterior Bulb (Motor neuron) Unknown or Redundant (Avery and Thomas, 1997) M2R Pharynx Posterior Bulb (Motor neuron) Unknown or Redundant (Avery and Thomas, 1997) MI Pharynx Anterior Bulb (Motor/ Intemeuron) Unknown or Redundant (Avery and Thomas, 1997) RIS Nerve Ring (Intemeuron) Unknown RMDDL Nerve Ring (Motor/ Intemeuron) Unknown RMDDR Nerve Ring (Motor/ Intemeuron) Unknown RMDL Nerve Ring (Motor/ Intemeuron) Unknown RMDR Nerve Ring (Motor/ Intemeuron) Unknown RMDVL Nerve Ring (Motor/ Interheuron) Unknown RMDVR Nerve Ring (Motor/ Intemeuron) Unknown SIADL Nerve Ring (Intemeuron) Unknown SIADR Nerve Ring (Intemeuron) Unknown SIAVL Nerve Ring (Intemeuron) Unknown SIAVL Nerve Ring (Intemeuron) Unknown SMDDL Nerve Ring (Motor/ Intemeuron) Unknown SMDDR Nerve Ring (Motor/ Intemeuron) Unknown SMDVL Nerve Ring (Motor/ Intemeuron) Unknown SMDVR Nerve Ring (Motor/ Intemeuron) Unknown ASs VNC (Motor neuron) Innervate Dorsal Muscle (Backward Locomotion) (Chalfie and White, 1988) DAs VNC (Motor neuron) Innervate Dorsal Muscle (Backward Locomotion) (Chalfie and White, 1988) DBs VNC (Motor neuron) Innervate Dorsal Muscle (Forward Locomotion) (Chalfie and White, 1988) VAs VNC (Motor neuron) Innervate Ventral Muscle (Backward Locomotion) (Chalfie and White, 1988) VBs VNC (Motor neuron) Innervate Ventral Muscle (Forward Locomotion) (Chalfie and White, 1988) SDQL Posterior Lateral (Intemeuron) Unknown DVB Dorso-Rectal Ganglia (Motor/ Intemeuron) Defecation (controls Emc) (Mclntireetal., 1993b) (Avery and Thomas, 1997) ALNL Lumbar Ganglia (Intemeuron) Unknown ALNR Lumbar Ganglia (Intemeuron) Unknown PLNL Lumbar Ganglia (Intemeuron) Unknown PLNR Lumbar Ganglia (Intemeuron) Unknown 166 construct was also expressed in class A, B and AS cholinergic motor neurons of the dorsal and ventral nerve cords and in neurons in the pre-anal, dorso-rectal (DVB), and lumbar ganglia (ALNL/R and PLNL/R) in the tail. A more detailed list of the neurons that have so far been identified to express the p«ca-7::GFP reporter construct is presented in Table 6. Although the pnca-7::GFP reporter construct was primarily localized within the nervous system its expression was not seen in any of the sensory cells. Therefore, GFP fluorescence was not observed in any of the chemosensory or mechanosensory neurons (ALM, PLM, A V M and PVD) nor was there any GFP expression observed in the command interneurons (AVA, AVB, AVD, AVE and PVC) responsible for generating locomotion in C. elegans. Likewise, there was no GFP expression observed in either the hypodermal or body wall muscle cells. nca-2 Gene Expression Pattern To examine the cellular expression pattern of the nca-2 gene, the nca-2 promoter: :GFP reporter construct (p/?ca-2::GFP) was generated in a multi-step process that involved piecing together four different PCR products. The first of these PCR products, clone "1", was generated by two rounds of PCR using the primer set KH91-KH94 and N2 genomic DNA for the first round and the nested primer set KH92-KH99. Two of the other PCR products, clones "2" and "3" were generated by PCR using the primer sets KH98-KH101 and KH93-KH100, respectively and the KH91-KH94 reaction as a template. The fourth PCR product, clone "C27F2.3 PCR" was generated by two rounds of PCR using the primer set KH79OS-KH80OS and N2 genomic DNA for the first round and the nested primer set KH79-KH80. All four of the PCR products were pieced together using common unique restriction enzymes. Thepnca-2::GFP reporter construct contained 3345 bp of putative promoter region upstream of the start site in exon la and had GFP fused in-frame with respect to the nca-2 open reading frame at exon 3 (Figure 1 IB). 167 The pnca-2::GFP reporter construct and the co-injection marker plasmid plin-15(+) were injected into the syncytial gonads of MT8189 /w-/5(«7c>5^Xhermaphrodites. Transgenic lines were subsequently established and the strain, TS174 vaExl8 was chosen as the source strain for integration of the extrachromosomal array. After integration, the transgenic strain TS191 valsl5 was examined for GFP expression. GFP fluorescence was found to be quite prevalent within the nervous system (Figure 27). Expression was observed late in embryogenesis and continued throughout development to the adult stage. The p«ca-2::GFP reporter construct was expressed in a number of neurons in the pharyngeal nervous system including the II interneurons (I1L/R) and the M4 motor neuron (data not shown). GFP expression was also observed in numerous neurons in the nerve ring of which only a few have been tentatively identified. The pnca-2:.G¥P reporter construct was also expressed in class A, B and AS cholinergic motor neurons and in class D GABAergic motor neurons of the dorsal and ventral nerve cords. In addition, the pnca-2::GFP reporter construct was also observed in neurons the dorso-rectal ganglion in the tail (DVA, DVB and DVC) and in the anal depressor muscle. A more detailed list of the neurons that have so far been identified to express the p«ca-2::GFP reporter construct is presented in Table 7. Although the pnca-2::G¥P reporter construct was primarily localized within the nervous system its expression was also observed in some non-neuronal tissues. For example, GFP expression was observed in some of sheath cells in the head (data not shown), in the vulval muscles (Figure 27) in the distal tip cells of the gonad (data not shown), as well as in the cells of the intestine (Figure 27). Similar to that observed for nca-1, the pnca-2 :.G¥P reporter construct was not expressed in the command interneurons (AVA, AVB, AVD, AVE and PVC) that are responsible for generating locomotion in C. elegans. Likewise, there was no GFP expression observed in the body wall muscle cells. 168 Figure 27. Expression of the nca-2 promoter: :GFP Fusion Construct (p«ca-2::GFP) in Transgenic Animals In all panels, the anterior of the animal is positioned to the left and the dorsal side is topmost. Images are of TS 191 valsl 5 transgenic animals expressing the pnca-2::GFP fusion construct. A) The overall GFP fluorescence pattern exhibited by a typical L4 hermaphrodite (left side view). GFP expression is observed in a few of the neurons in the pharyngeal nervous system, as well as in numerous unidentified nerve ring interneurons {filled arrowhead). GFP expression is also observed in the majority of the motor neuron cell bodies present in the ventral nerve cord (VNC, double arrowhead), in the vulval muscles (VM, *) and in neurons and muscle cells in the tail (arrow). Diffuse GFP expression is also observed in the intestine (INT). Scale bar = 50 um. B) The GFP fluorescence pattern exhibited in the ventral nerve cord of a typical L4 hermaphrodite around the hypodermal cells P9 and P10 (left side view). The identities of the cells expressing GFP are labeled in panel C for reference. GFP expression is extremely faint in the GABAergic motor neurons DD5, VD10 (not in focus) and VD11. GFP expression is absent in DA7 in this animal due to mosaic expression in the TS 191 valsl5 strain. Scale bar = 10 um. C) Merged DIC and GFP fluorescent images of the same animal in panel B. GFP expression is observed in the class A (VA10 and VA11), class B (VB11 and DB7) and class AS (AS10) cholinergic motor neuron, as well as in the class D (DD5, VD10 and VD11) GABAergic motor neurons. Scale bar = 10 um. D) The GFP fluorescence pattern exhibited in the tail region of a typical L4 hermaphrodite (left side view). GFP expression is observed in the DVB neuron required for defecation, in neuron DVC, as well as in the anal depressor muscle (ADM). DVA is out of the plane of focus. Scale bar =10 um. E) Merged DIC and GFP fluorescent images of the same animal in panel D. Scale bar = 10 um. 169 170 Table 7. Summary of Tentatively Identified Neurons Expressing the pnca-2::GFP Reporter Construct Cell Location (Cell Type) Function Reference I1L Pharynx Anterior Bulb (Intemeuron) Connected to Extraphyngeal Nervous System via RIPs Modulates Pumping Rate in Absence of Bacteria (Avery and Thomas, 1997) I1R Pharynx Anterior Bulb (Intemeuron) Connected to Extraphyngeal Nervous System via RIPs Modulates Pumping Rate in Absence of Bacteria (Avery and Thomas, 1997) M4 Pharynx Anterior Bulb (Motor neuron) Posterior Isthmus Peristalsis (Avery and Thomas, 1997) RIML Nerve Ring (Motor neuron) Unknown RIMR Nerve Ring (Motor neuron) Unknown RMDDL Nerve Ring (Motor/ Intemeuron) Unknown RMDDR Nerve Ring (Motor/ Intemeuron) Unknown RMDL Nerve Ring (Motor/ Intemeuron) Unknown RMDR Nerve Ring (Motor/ Intemeuron) Unknown RMDVL Nerve Ring (Motor/ Intemeuron) Unknown RMDVR Nerve Ring (Motor/ Intemeuron) Unknown SIADL Nerve Ring (Intemeuron) Unknown SIADR Nerve Ring (Intemeuron) Unknown SIAVL Nerve Ring (Intemeuron) Unknown SIAVL Nerve Ring (Intemeuron) Unknown SMDDL Nerve Ring (Motor/ Intemeuron) Unknown SMDDR Nerve Ring (Motor/ Intemeuron) Unknown SMDVL Nerve Ring (Motor/ Intemeuron) Unknown SMDVR Nerve Ring (Motor/ Intemeuron) Unknown ASs VNC (Motor neuron) Innervate Dorsal Muscle (Backward Locomotion) (Chalfie and White, 1988) DAs VNC (Motor neuron) Innervate Dorsal Muscle (Backward Locomotion) (Chalfie and White, 1988) DBs VNC (Motor neuron) Innervate Ventral Muscle (Forward Locomotion) (Chalfie and White, 1988) DDs VNC (Motor neuron) Innervate Dorsal Muscle (Muscle Inhibition) (Driscoll and Kaplan, 1997) VAs VNC (Motor neuron) Innervate Ventral Muscle (Backward Locomotion) (Chalfie and White, 1988) VBs VNC (Motor neuron) Innervate Dorsal Muscle (Forward Locomotion) (Chalfie and White, 1988) VDs VNC (Motor neuron) Innervate Ventral Muscle (Muscle Inhibition) (Driscoll and Kaplan, 1997) DVA Dorso-Rectal Ganglia (Ring/ Intemeuron) Mechanosensory integration (Wicks et al., 1996) (Driscoll and Kaplan, 1997) DVB Dorso-Rectal Ganglia (Motor/ Intemeuron) Defecation (controls Emc) (Mclntire et al., 1993b) (Avery and Thomas, 1997) DVC Dorso-Rectal Ganglia (Ring/ Intemeuron) Unknown j i 171 Coexpression of the nca-1 and nca-2 Genes In order to confirm the observation that nca-1 and nca-2 are coexpressed in the cholinergic motor neurons of the dorsal and ventral nerve cords and in the GABAergic neuron DVB, the transgenic strain TS 199 valsll; valsl5 was generated. The opening reading frame for GFP in the pnca-1 ::GFP reporter construct was replaced with that of DsRed2 and was injected into the syncytial gonad of MT8189 /w-75(«76\5^Xhermaphrodites along with the co-injection marker plasmid plin-15(+). Transgenic lines were subsequently established and after integration produced the transgenic strain TS164 valsll. The DsRed2 expression pattern exhibited by the newly generated TS164 valsll transgenic strain was identical to the GFP expression pattern of the TS48 vals6 transgenic strain. Using classical genetic techniques, the transgenic strain TS 199 valsll; valsl5 was generated from TS164 valsll and TS191 valsl5. Examination of the GFP and DsRed2 fluorescence patterns confirmed that the coexpression of the p«ctf-7::DsRed2 and pnca-2::GYP reporter constructs in the cholinergic motor neurons of the dorsal and ventral nerve cords (Figure 28) and in the GABAergic neuron DVB (Figure 29). The results also confirmed that only the pnca-2::GFP reporter construct is expressed in GABAergic motor neurons of the dorsal and ventral nerve cords (Figure 28). Phenotypic Analysis of nca Mutant Strains Locomotion Based upon the observation that the individual nca-1 and nca-2 gene knockout strains did not exhibit any obvious visible phenotype and that both the pnca-1 and pnca-2:.G¥? reporter constructs are expressed in cholinergic motor neurons of the dorsal and ventral nerve cords (Figures 26,27 and 28), it was hypothesized that there may be some functional interaction and/or redundancy between nca-1 and nca-2 and that combined mutations in both of these genes may 172 Figure 28. Coexpression of the nca-1 promoter: :DsRed2 Fusion Construct and the nca-2 promoter: :GFP Fusion Construct in the Ventral Nerve Cord In all panels, the anterior of the animal is positioned to the left and the dorsal side is topmost. Images are of TS 199 valsll; valsl5 transgenic animals expressing both the p«ca-7::DsRed2 and pnca-2::GFP fusion constructs. The GFP exposure was increased to detect the low levels of expression in the GABAergic motor neurons. Some bleed through of DsRed2 is present in the GFP images. GFP expression is largely cytosolic, whereas DsRed2 expression is entirely nuclear. A) The GFP fluorescence pattern exhibited in the ventral nerve cord of a typical L4 hermaphrodite around the hypodermal cells P9 and P10 (left side view). B) The DsRed2 fluorescence pattern exhibited by the same animal as in panel A. C) Merged GFP and DsRed2 fluorescent images. GFP and DsRed2 coexpression is observed in the class A (VA10), class B (VB11 and DB7) and class AS (AS 10) cholinergic motor neurons. DsRed2 is not expressed in the class D (VD10 and VD11) GABAergic motor neurons, whereas GFP expression is observed in these cells. D) Merged DIC, GFP and DsRed2 images. Scale bar = 10 um. 173 174 Figure 29. Coexpression of the nca-1 promoter::DsRed2 Fusion Construct and the nca-2 promoter::GFP Fusion Construct in the GABAergic Neuron DVB In all panels, the anterior of the animal is positioned to the left and the dorsal side is topmost. Images are of TS 199 valsll; valsl5 transgenic animals expressing both the p«ca-/::DsRed2 and pnca-2::GFP fusion constructs. Some bleed through of DsRed2 is present in the GFP images. GFP expression is largely cytosolic, whereas DsRed2 expression is entirely nuclear. A) The GFP fluorescence pattern exhibited in the tail region of a typical L4 hermaphrodite. GFP expression in DVB is indicated by an arrow (left side view). B) The DsRed2 fluorescence pattern exhibited by the same animal as in panel A. C) Merged GFP and DsRed2 fluorescent images. GFP and DsRed2 coexpression is observed in the GABAergic neuron DVB. D) Merged DIC, GFP and DsRed2 images. Scale bar = 10 um. 175 176 lead to the generation of a locomotion phenotype. In order to address the question of whether there may be some functional interaction and/or redundancy between nca-1 and nca-2, a double mutant between nca-1(gk.9) and nca-2(gk5) was generated. The resulting TS65 nca-2(gk5)III; nca-1 (gk9)IV double mutant strain was examined for any visible mutant phenotypes. The double mutant exhibited an obvious uncoordinated locomotion phenotype suggesting that there may be functional redundancy between nca-1 and nca-2 in the cholinergic motor neurons of the dorsal and ventral nerve cords. The uncoordinated movement exhibited by the nca-2(gk5); nca-1 (gk9) mutant consisted of cycling between periods of inactivity (lasting up to 10 s) and episodes of either forward or backward movement (rarely more than a few body lengths) suggesting that in this double mutant there may be a defect in at least some of the neurons responsible for generating the locomotion program in C. elegans. Body Bends and Thrashing For a more quantitative assessment of the locomotion defect exhibited by the nca-2(gk5); nca-1(gk9) double mutant strain both the body bends and thrashing assays were used. In these assays, the number of bends at the mid-body over a specified period of time for animals on plates or in liquid were countered, respectively (Miller et al., 1996; Robatzek and Thomas, 2000; Tsalik and Hobert, 2003). The number of body bends in a 30 s interval was not significantly different (P > 0.05) from wild-type (36.7 +/- 0.5; n = 10) for either the nca-l(gk9) (35.6 +/- 0.9; n = 10) or nca-2(gk5) (36.5 +/-1.1; n = 10) single mutant strains. In contrast, the number of body bends was significantly different (P < 0.05) from wild-type (36.7 +/- 0.5; n = 10) for the nca-2(gk5); nca-1 (gk9) (10.9 +/-1.3; n = 10) double mutant strain (Figure 30A; Table 8). Similarly, the number of thrashes/min was not significantly different (P > 0.05) from wild-type (185.9 +/- 6.8; n = 10) for either the nca-l(gk9) (190.5 +/- 8.0; n = 20) or nca-2(gk5) (177.8 +/- 9.4; n = 10) single mutant strains. In contrast, the number of thrashes was significantly different (P < 0.05) 177 Figure 30. Body Bends and Thrashing Analysis of nca Mutant Strains A) The body bends analysis of the nca-1 (gk9), nca-2(gk5) and nca-2(gk5); nca-1 (gk9) mutant strains. Bars represent the average of data collected from 10 animals from each strain over a 30 s interval and represent mean +/- SEM. Only the nca-2(gk5); nca-1 (gk9) double mutant strain was significantly different from wild-type (P < 0.05, One-way ANOVA, Origin) and is indicated by an *. The nca-2(gk5); nca-1 (gk9) double mutant strain was also significantly different from both the nca-1 (gk9) and nca-2(gk5) mutant strains (P < 0.05, One-way ANOVA, Origin) and there was no significant difference between the nca-1 (gk9) and nca-2(gk5) mutant strains (P > 0.05, One-way ANOVA, Origin). B) The thrashing analysis of the nca-1(gk9), nca-2(gk5) and nca-2(gk5); nca-l(gk9) mutant strains. Bars represent the average of data collected from 10 (N2 and nca-2(gk5)) or 20 (nca-l(gk9) and nca-2(gk5); nca-l(gk9)) animals from each strain over a one min interval and represent mean +/- SEM. Only the nca-2(gk5); nca-1 (gk9) double mutant strain was significantly different from wild-type (P < 0.05, One-way ANOVA, Origin) and is indicated by an *. The nca-2(gk5); nca-1(gk9) double mutant strain was also significantly different from both the nca-1 (gk.9) and nca-2(gk5) mutant strains (P < 0.05, One-way ANOVA, Origin) and there was no significant difference between the nca-1 (gk9) and nca-2(gk5) mutant strains (P > 0.05, One-way ANOVA, Origin). 178 A 179 Table 8. Summary of the Body Bends and Thrashing Rates for nca Mutant Strains Strain Body Bends/30s Thrashes/min wild-type 36.7+/-0.5(n=10) 185.9+/-6.8 (n= 10) nca-1 (gk.9) 35.6+/-0.9 (n=10) 190.5+/-8.0 (n = 20) nca-2(gk5) 36.5+/-l.l(n=10) 177.8+/-9.4 (n=10) nca-2(gk5); nca-1(gk9) 10.9+/- 1.3(n=10)* 10.5+/-1.4 (n = 20)* values are mean +/- SEM * significantly different from wild-type (P < 0.05, One-way ANOVA, Origin) 181 from wild-type (185.9 +/- 6.8; n = 10) for the nca-2(gk5); nca-1 (gk9) (10.5 +/-1.4; n = 20) double mutant strain (Figure 30B; Table 8). The results of the body bends and thrashing assays confirmed the notion that there may be a functional interaction and/or redundancy between nca-1 and nca-2 in the cholinergic motor neurons of the dorsal and ventral nerve cords. The data also suggested that the NCA-1 and NCA-2 ion channels may contribute to the electrical properties of motor neurons and/or to synaptic transmission itself. Aldicarb and Nicotine Assays The class A, B and AS motor neurons that synapse onto the dorsal and ventral body wall muscles in C. elegans are cholinergic, releasing acetylcholine (ACh) at the neuromuscular junction (NMJ) (Alfonso et al., 1993; Driscoll and Kaplan, 1997). Upon release, ACh diffuses across the synaptic cleft at the NMJ and binds to post-synaptic nicotinic ACh receptors on the body wall muscle which in turn causes the muscle to depolarize and to contract. Body wall muscle contraction is terminated by the cleavage of ACh into acetyl CoA and choline by the enzyme acetylcholinesterase (AChE). Inhibitors of AChE, such as aldicarb, prevent the breakdown of ACh in the synaptic cleft of the NMJ by inhibiting the activity of AChE. In the presence of aldicarb, ACh levels accumulate at the NMJ and causes an excessive stimulation of the body wall muscles leading to hypercontraction, paralysis and eventual death in wild-type animals. Previous genetic screens have identified mutations that can confer resistance to the effects of the AChE inhibitors (Brenner, 1974; Nguyen et al., 1995; Miller et al., 1996). Mutant strains that are resistant to AChE inhibitors do not hypercontract to the same extent as wild-type animals and are capable of living and reproducing in the presence of the drug. Furthermore, the degree of resistance conferred by any given mutation to AChE inhibitors is directly related to the amount of ACh that is released at the NMJ and/or the sensitivity of the body wall muscles to the presence of ACh (Miller et al., 1996; Rand and Nonet, 1997). Mutations that confer aldicarb 182 resistance fall generally into two different classes. The first class of aldicarb resistant mutants consists of mutations that decrease the amount of ACh that is released from cholinergic motor neurons at the NMJ, whereas the second class of aldicarb resistant mutants consists of mutations that decrease the sensitivity of the body wall muscle to ACh. The two classes of aldicarb resistant mutants can be distinguished based upon their sensitivity to agonists of the nicotinic ACh receptor, such as nicotine and levamisole. Exogenous application of either nicotine or levamisole causes hypercontraction and death in wild-type animals due to excessive stimulation of the body wall muscles. Taken together, mutants that have defects in pre-synaptic release of ACh are resistant to aldicarb but are sensitive to the presence of nicotine (Rand and Nonet, 1997). In contrast, mutants that have defects in the post-synaptic sensitivity to ACh are resistant to both aldicarb and nicotine (Miller et al., 1996). In order to determine whether the locomotion phenotype exhibited by the nca-2(gk5); nca-l(gk9) double mutant strain could, in part, be due to a cholinergic defect at the NMJ animals were examined with both the aldicarb and nicotine assays. Figure 31 and Table 9 show that the nca-2(gk5); nca-1 (gk.9) double mutant was significantly resistant to aldicarb (time at which 50% of animals were paralyzed = 104.9 +/- 4.4 min; n = 2 trials) (P < 0.05) as compared to wild-type (time at which 50% of animals were paralyzed = 85.0 +/-1.3 min; n = 2 trials). Both the nca-l(gk9) (time at which 50% of animals were paralyzed = 90.3 +/-1.2 min; n = 2 trials) and nca-2(gk5) (time at which 50% of animals were paralyzed = 88.6 +/-1.5 min; n = 2 trials) mutant strains exhibited wild-type sensitivity to aldicarb (P > 0.05). The results of the aldicarb assay indicated that only the nca-2(gk5); nca-1 (gk9) double mutant strain was more resistant to aldicarb as evident by the delay in the onset of paralysis. In addition, all four strains were exposed to a solution of 1% nicotine and examined after 10 min to access their sensitivity to this drug. After approximately 10 minutes, all four mutant strains had hypercontracted in the presence of 1% nicotine and were dead within a half an hour (Table 9). Taken together, the 183 Figure 31. Percentage of nca Mutant Animals Paralyzed Over Time on 1 mM Aldicarb The percentage of animals paralyzed is plotted versus time to give an estimate of aldicarb resistance. Data points represent mean +/- SEM for two trials consisting of 20 animals per trial and the time at which 50% of animals were paralyzed for each strain is represented as mean +/-SEM. The nca-2(gk5); nca-1 (gk9) double mutant strain (time at which 50% of animals were paralyzed = 104.9 +/- 4.4 min) was resistant to aldicarb (P < 0.05, Allfit) when compared to wild-type (time at which 50% of animals were paralyzed = 85.0 +/-1.3 min) as evident by the slow rate of onset of paralysis, whereas the nca-1 (gk9) (time at which 50% of animals were paralyzed = 90.3 +/-1.2 min) and nca-2(gk5) (time at which 50% of animals were paralyzed = 88.6 +/-1.5 min) mutant strains exhibited a wild-type rate of onset of paralysis (P > 0.05, Allfit). 184 Minutes on 1 mM Aldicarb 185 Table 9. Summary of the Aldicarb, Nicotine and Halothane Assays Strain 1 mM aldicarb w 1% Nicotine Halothane (EC 5 0 ) wild-type 85.0+/-1.3 min hypercontract 0.56 +/- 0.06 vol% nca-1 (gk9) 90.3 +/-1.2 min hypercontract 0.69 +/- 0.05 vol% nca-2(gk5) 88.6+/-1.5 min hypercontract 0.48 +/- 0.04 vol% nca-2(gk5); nca-l(gk9) 104.9 +/- 4.4 min* hypercontract 0.28 +/- 0.07 vol%* values are mean +/- SEM for two trials * significantly different from wild-type (P < 0.05, Allfit) vi/ time at which 50% of the animals were paralyzed 186 results of the aldicarb and nicotine assays suggest that the locomotion phenotype exhibited by the nca-2(gk5); nca-1 (gk9) double mutant strain may be partially due to a pre-synaptic defect in ACh release at the NMJ and not due to a defect in post-synaptic function. This result agrees with the promoter::GFP expression data for nca-1 and nca-2, as both the pnca-1 and pnca-2::GFP reporter constructs were found to be expressed in cholinergic motor neurons of the dorsal and ventral nerve cords and neither reporter construct was expressed in body wall muscle. Halothane Assay To further confirm that the locomotion defect exhibited by the nca-2(gk5); nca-1 (gk.9) double mutant strain was due to a pre-synaptic defect in ACh release at the NMJ, animals were examined using the halothane assay. Halothane is a volatile anesthetic that blocks memory formation, consciousness and voluntary movement in humans, and is used as a surgical anesthetic. While its mechanism of action is not well understood, previous work on the effect of halothane on glutamate-mediated synaptic transmission at the NMJ in Drosophila larvae has shown that in the presence of halothane, there is a marked reduction in the amplitude of nerve-evoked excitatory junctional currents (EJCs), suggesting that at least one halothane-mediated effect is to depress pre-synaptic neurotransmission at the NMJ (Nishikawa and Kidokoro, 1999). Of particular relevance, mutants with altered sensitivities to halothane show different levels of pre-synaptic depression (Nishikawa and Kidokoro, 1999) and the Drosophila nca homologue Dmaiu (also called har38 and har85) was first identified in a screen for altered halothane sensitivity (Krishnan and Nash, 1990; Nash et al., 2002). Similarly, it has been found that in C. elegans the predominant mechanism for the effect of halothane on the NMJ and locomotion appears to be a pre-synaptic mechanism (van Swinderen et al., 1999). Previous work on the effects of halothane on C. elegans has shown that mutations in genes encoding synaptic transmitter release proteins such as unc-64 (syntaxin) (Saifee et al., 1998) and ric-4 (SNAP-25) 187 (Nguyen et al., 1995; Miller et al., 1996) have altered sensitivity to halothane due to a changes in cholinergic neurotransmission at the NMJ (van Swinderen et al., 1999; van Swinderen et al., 2001). In general, for C. elegans it has been found that mutants that are resistant to the AChE inhibitor, aldicarb, are also hypersensitive to halothane and that mutants that are hypersensitive to aldicarb are resistant to halothane (van Swinderen et al., 2001). Based upon these findings on the effect of halothane at the NMJ in Drosophila and C elegans, it was hypothesized that the pre-synaptic cholinergic defect present in the nca-2(gk5); nca-1 (gk9) double mutant strain may be enhanced in the presence of halothane and result in a hypersensitive phenotype. Figure 32 and Table 9 show that the nca-2(gk5); nca-1 (gk9) double mutant was significantly hypersensitive (EC 5 0 = 0.28 +/- 0.07 vol%; n = 2 trials) (P < 0.05) to halothane as compared to wild-type (EC50 = 0.56 +/- 0.06 vol%; n = 2 trials), whereas the nca-l(gk9) (EC 5 0 = 0.69 +/- 0.05 vol%; n = 2 trials) and nca-2(gk5) (EC 5 0 = 0.48 +/- 0.04 vol%; n = 2 trials) mutant strains were not significantly different from wild-type (EC50 = 0.56 +/- 0.06 vol%; n = 2 trials) (P > 0.05). Taken together, these results further suggest that the nca-2(gk5); nca-l(gk9) double mutant strain exhibits a pre-synaptic cholinergic defect. Interactions Between nca-1, nca-2 and the unc-2 C a 2 + Channel Gene The enhancement of the locomotion defect exhibited by the nca-2(gk5); nca-(gk9) double mutant strain in the presence of halothane lead to the hypothesis that there may be genetic or functional interactions between nca-1 and nca-2 and other genes involved in the pre-synaptic release. In order to address this hypothesis, a series of double and triple mutant strains were generated with nca-1 and/or nca-2 and either reduction-of function or loss-of-function alleles of unc-2. The unc-2 gene in C. elegans encodes for a non L-type voltage-gated Ca 2 + channel (Schafer and Kenyon, 1995) which is believed to be the primary Ca 2 + channel involved in triggering neurotransmitter release at the NMJ (Richmond et al., 2001; Mathews et al., 2003). 188 Figure 32. Halothane Concentration-Response Curves for the nca Mutant Strains The radial dispersal index, the fraction of animals moving in 40 min from the center to the bacterial ring at the edge of the assay plate, is plotted against halothane concentration to estimate sensitivity to the anesthetic (n = two trials). The EC50 of the nca-2(gk5); nca-1 (gk9) double mutant (0.28 +/- 0.07 vol%; mean +/- SEM) was significantly hypersensitive (P < 0.05, Allfit) as compared to wild-type (0.56 +/- 0.06 vol%), whereas the EC 5 0's of the nca-1 (gk9) (0.69 +/- 0.05 vol%) and nca-2(gk5) (0.48 +/- 0.04) mutant strains were not significantly different (P > 0.05, Allfit) from wild-type (0.56 +/- 0.06 vol%). The nca-2(gk5); nca-1 (gk.9) double mutant strain was also significantly different from both the nca-1 (gk.9) and nca-2(gk5) mutant strains (P < 0.05, Allfit) and there was no significant difference between the nca-1 (gk9) and nca-2(gk5) mutant strains (P > 0.05, Allfit). 189 [Halothane] vo!% 190 unc-2 mutants exhibit both locomotion defects and aldicarb resistance due to a pre-synaptic defect in the release of ACh (Miller et al., 1996; Richmond et al., 2001; Mathews et al., 2003). In addition, electrophysiological recordings from loss-of-function unc-2(e55) mutant animals show an ~3 fold decrease in evoked release at the NMJ (Richmond et al., 2001). Two different unc-2 alleles were used to examine whether there is a genetic interaction between nca-1, nca-2 and unc-2. The first of the unc-2 alleles used was the putative reduction-of-function ox8 allele. The ox8 allele is a R1284H missense mutation in the S4 region of domain IV (C. Thacker and T. Snutch, pers. comm.). The missense mutation in unc-2(ox8) is predicted to be a reduction-of-function mutation as the amino acid substitution in the voltage sensor of domain JV may alter the voltage-dependent properties of the UNC-2 Ca 2 + channel and decrease the amount of calcium influx through UNC-2 at the NMJ. Figure 33A and Table 10 show that the number of thrashes/min exhibited by the nca-2(gk5); nca-1(gk9) (10.5 +/-1.4; n = 20) double mutant strain was significantly less (P < 0.05) than that of unc-2(ox8) (37.6 +/- 2.9; n = 10) indicating that the locomotion defect of the double nca mutant is more severe than that of unc-2(ox8) as assessed by this assay. Furthermore, only when both the nca-1 (gk9) and nca-2(gk5) mutations were added together to the unc-2(ox8) mutant background did the number of thrashes/min of the unc-2 (ox8) ; nca-2(gk5); nca-1/g&9/mutant (5.0 +/- 0.5; n = 20) significantly decrease (P < 0.05). The results of the thrashing assay for the unc-2(ox8); nca mutants suggest that there may be a genetic interaction between nca-1, nca-2 and unc-2 as the locomotion defect was enhanced in the unc-2(ox8); nca-2 (gk5); nca-1 (gk.9) triple mutant. The second unc-2 allele examined was the putative loss-of function el29 allele. The el29 allele is a nonsense mutation in the S4 region of domain I and results in the substitution of R214 for a stop codon (C. Thacker and T. Snutch, pers. comm.). The early nonsense mutation in unc-2{el29) is predicted to be a loss-of-function mutation as the mutation is predicted to result in a prematurely truncated non-functional UNC-2 Ca 2 + channel and a loss of calcium influx through 191 Figure 33. Thrashing Analysis of unc-2; nca Mutant Strains A) The thrashing analysis of unc-2(ox8); nca mutant strains. Bars represent the average of data collected for each strain over a one minute interval and represent mean +/- SEM. Strains marked with an * were significantly different from wild-type (P < 0.05, One-way ANOVA, Origin), those strains marked with an v|/ were significantly different than unc-2(ox8) (P < 0.05, One-way ANOVA, Origin) and the strain marked with an § was significantly different from nca-2(gk5); nca-1 (gk9) (P < 0.05, One-way ANOVA, Origin). There was no significant difference between the strains unc-2(ox8), unc-2(ox8); nca-1(gk.9) and unc-2(ox8); nca-2(gk5) (P > 0.05, One-way ANOVA, Origin). B) The thrashing analysis of unc-2(el29); nca mutant strains. Bars represent the average of data collected for each strain over a one minute interval and represent mean +/- SEM. Strains marked with an * were significantly different from wild-type (P < 0.05, One-way ANOVA, Origin) but not from one another except the strain marked with an VJ was significantly different from all the other strains marked with an * (P < 0.05, One-way ANOVA, Origin). 192 A 200 n=20 .S 190 n=10 n=10 40 ^ 180-1 CD GO H 20-10 : 0 r ^ T sv-v o v ^ \ v 4* «p <^ <F <F ^ ^ & 193 194 Table 10. Summary of Thrashing Rates for Different unc-2(ox8); nca Mutant Strains Strain Thrashes/min wild-type 185.9 +/-6.8(n= 10) nca-1 (gk9) 190.5 +/- 8.0 (n = 20) nca-2(gk5) 177.8 +/- 9.4 (n= 10) nca-2(gk5); nca-1 (gk9) 10.5+/- 1.4(n = 20)* unc-2(ox8) 37.6+/-2.9 (n = 10)* unc-2(ox8); nca-l(gk9) 34.9+/-5.3 (n=10)* unc-2 (0x8); nca-2(gk5) 31.8 +/- 3.8 (n = l O W unc-2(ox8); nca-2(gk5); nca-l(gk9) 5.0+/-0.5 (n = 20V* u/d> values are mean +/- SEM *, v/, <|) significantly different from wild-type, unc-2(ox8) and nca-2(gk5); nca-1(gk9), respectively (P < 0.05, One-way ANOVA, Origin) 195 Table 11. Summary of Thrashing Rates for Different unc-2(el29); nca Mutant Strains Strain Thrashes/min wild-type 185.9 +/-6.8(n = 10) nca-1 (gk9) 190.5 +/- 8.0 (n = 20) nca-2(gk5) 177.8+/-9.4 (n= 10) nca-2 (gk5); nca-1(gk.9) 10.5+/- 1.4(n = 20)* unc-2(el29) 13.2 +/-2.1 (n= 10)* unc-2(el29); nca-1(gk9) 9.3+/- 1.0 (n= 18)* unc-2(el29); nca-2(gk5) 8.4 +/- 0.9 (n = 10)* unc-2(el29); nca-2(gk5); nca-1 (gk9) 1.6 +/-0.5(n = 9)*u; values are mean +/- SEM * significantly different from wild-type (P < 0.05, One-way ANOVA, Origin) vy significantly different from nca-2 (gk5); nca-1 (gk9) and unc-2 (el 29) (P < 0.05, One-way ANOVA, Origin) 196 UNC-2 at the NMJ. Figure 33B and Table 11 show that the number of thrashes/min exhibited by the nca-2(gk5); nca-1 (gk9) (10.5 +/-1.4; n = 20) double mutant strain was not significantly different (P > 0.05) than that of unc-2(el29) (13.2 +/- 2.1; n = 10). Furthermore, only when both the nca-1(gk.9) and nca-2(gk5) mutations were added to the unc-2(el29) mutant background did the number of thrashes/min of the unc-2(el29); nca-2(gk5); nca-1(gk9) mutant (1.6 +/- 0.5; n = 9) significantly decrease (P < 0.05). The results of the thrashing assay for the unc-2(el29); nca mutants raise the possibility that nca-1 and nca-2 may interact with another Ca 2 + channel in ACh release at the NMJ as the locomotion defect of the unc-2 (el 29); nca-2 (gk5); nca-1 (gk9) triple mutant was enhanced despite the predicted absence of UNC-2 channels in this mutant strain. Interactions Between nca-1, nca-2, cca-1 and unc-2 VGIC Genes The enhanced locomotion defect observed for the unc-2 (el 29); nca-2 (gk5); nca-1 (gk9) triple mutant raised the possibility that an additional Ca 2 + channel to UNC-2 may be involved in ACh release at the NMJ. This possibility agrees with the finding that loss-of-function unc-2(e55) mutant animals retain a significant amount of evoked release presumably due to additional sources of Ca 2 + entry at the NMJ (Richmond et al., 2001). In order to test this hypothesis, a number of different mutants between nca-1, nca-2 and cca-\ were generated. The cca-1 gene in C. elegans encodes for a T-type Ca 2 + channel (Cribbs et al., 1998; Perez-Reyes et al., 1998; Lee et al., 1999b; McRory et al., 2001) and is expressed in a subset of motor neuron cell bodies in the ventral nerve cord (C. Thacker and T. Snutch, pers. comm.). The cca-1 allele examined was the putative loss-of function adl650 allele. Molecular analysis has determined that the mutation in the adl650 allele is an -2.5 kb deletion that removes exons encoding the last half of domain II and the majority of domain III and alters the reading frame (C. Thacker and T. Snutch, pers. comm.). Figure 34 and Table 12 show that the number of thrashes/min for the cca-1 (adl650); nca-2(gk5); nca-1 (gk9) triple mutant strain (8.6 +/- 0.8; n = 20) was significantly different 197 Figure 34. Thrashing Analysis oi cca-l(adl650);nca Mutant Strains The thrashing analysis of the different cca-1 (adl650); nca mutant strains. Bars represent the average of data collected for each strain over a one minute interval and represent mean +/- SEM. Strains marked with an * were significantly different from wild-type (P < 0.05, One-way ANOVA, Origin). All other strains were not significantly different from one another (P > 0.05, One-way ANOVA, Origin). 198 199 Table 12. Summary of Thrashing Rates for Different cca-1(adl650); nca Mutant Strains Strain Thrashes/min wild-type 185.9 +/-6.8(n= 10) nca-l(gk9) 190.5 +/- 8.0 (n = 20) nca-2(gk5) 177.8+/-9.4 (n= 10) nca-2 (gk5); nca-1 (gk9) 10.5+/-1.4 (n = 20)* cca-l(adl650) 178.3+/-8.8 (n= 10) cca-1 (adl650); nca-l(gk9) 175.4+/-8.7 (n= 10) cca-1 (adl650); nca-2(gk5) 196.0+/-10.1 (n= 10) cca-1 (adl650); nca-2(gk5); nca-1 (gk9) 8.6 +/- 0.8 (n = 20)* values are mean +/- SEM * significantly different from wild-type (P < 0.05, One-way ANOVA, Origin) 200 (P < 0.05) from wild-type (36.7 +/- 0.5; n = 10) and that all other cca-1 (adl650) mutant strains were not significantly different (P > 0.05). Furthermore, the number of thrashes/min for the cca-l(ad!650); nca-2(gk5); nca-1 (gk9) triple mutant strain (8.6 +/- 0.8; n = 20) was not significantly different (P > 0.05) from nca-2(gk5); nca-1 (gk9) (10.5 +/-1.4). In addition, Figure 35 and Table 13 show that the number of thrashes/min of the unc-2(ox8) cca-1 (adl650); nca-2(gk5); nca-1 (gk9) mutant strain (3.8 +/- 0.8; n = 10) strain was not significantly different (P > 0.05) from unc-2(ox8); nca-2(gk5); nca-1(gk.9) mutant strain (5.0 +/-0.5; n = 20), but was significantly higher (P < 0.05) than the unc-2(el29); nca-2(gk5); nca-l(gk9) mutant strain (1.6 +/- 0.5; n = 9). The results of the cca-1 (adl650) mutant strains show that the cca-1 gene does not seem to interact with either nca-1 or nca-2 to alter ACh release at the NMJ (Figure 34). Furthermore, the inability of the unc-2(ox8) cca-1 (adl 650); nca-2(gk5); nca-1(g£9)mutant strain to approach the locomotion defect observed for the unc-2(el29); nca-2(gk5); nca-1 (gk.9) triple mutant suggests that cca-1 is unlikely to make a significant contribution to ACh release at the NMJ (Figure 35). Interactions Between nca-1, nca-2 and unc-25 In order for C . elegans to generate sinusoidal movement, dorsal and ventral body wall muscle contraction must be out of phase. This is achieved by the coordinated excitation of body wall muscles on one side of the animal by the excitatory class A or B cholinergic motor neurons (Alfonso et al., 1993; Driscoll and Kaplan, 1997) and the simultaneous cross-inhibition of the body wall muscles on the opposite side by the class D inhibitory GABAergic motor neurons (Chalfie and White, 1988; Mclntire et al., 1993b). Support for the role of the class D motor neurons in cross-inhibition comes from laser ablation studies wherein destruction of the VD and DD motor neurons caused a "shrinking" movement phenotype due to the simultaneous 201 Figure 3 5 . Thrashing Analysis of VGIC Mutant Strains The thrashing analysis of VGIC mutant strains. Bars represent the average of data collected for each strain over a one minute interval and represent mean +/- SEM. All strains were significantly different from wild-type (P < 0.05, One-way ANOVA, Origin). There was no significant difference between nca-2(gk5); nca-1(gk9) and cca-1 (adl650); nca-2(gk5); nca-l(gk9) (P > 0.05, One-way ANOVA, Origin). Strains marked with an * were significantly different from nca-2(gk5); nca-1 (gk9) and cca-1 (adl650); nca-2(gk5); nca-1(gk.9) (P < 0.05, One-way ANOVA, Origin), but not from one another. The strain marked with a i|/ was significantly different from unc-2(ox8); nca-2(gk5); nca-1(gk.9) and unc-2(ox8) cca-1 (adl650); nca-2(gk5); nca-1 (gk9) (P < 0.05, One-way ANOVA, Origin). 202 Table 13. Summary of Thrashing Rates for Different VGIC Mutant Strains Strain Thrashes/min wild-type 185.9+/-6.8 (n= 10) nca-2(gk5); nca-l(gk9) 10.5+/- 1.4 (n = 20) cca-1 (adl650); nca-2(gk5); nca-1(gk9) 8.6+/-0.8(n = 20) unc-2 (0x8); nca-2 (gk5); nca-l(gk9) 5.0+/-0.5(n = 20)* unc-2(ox8) cca-1 (adl650); nca-2(gk5); nca-1 (gk9) 3.8+/-0.8 (n= 10)* unc-2(el29); nca-2(gk5); nca-1 (gk9) 1.6+/-0.5(n = 9V*vt/ values are mean +/- SEM * significantly different from nca-2(gk5); nca-1 (gk9) and cca-1 (adl650); nca-2(gk5); nca-1 (gk9) (P < 0.05, One-way ANOVA, Origin) 11/ significantly different from unc-2(ox8); nca-2(gk5); nca-l(gk9) and unc-2(ox8) cca-1(adl650); nca-2(gk5); nca-1(gk9) (P < 0.05, One-way ANOVA, Origin) 204 hypercontraction of dorsal and ventral body wall muscles (Mclntire et al., 1993b). Furthermore, mutations in genes involved in the development and function of the class D motor neurons, such as the glutamic acid decarboxylase gene unc-25, also cause the "shrinker" movement phenotype (Mclntire et al., 1993a; Jin et al., 1999). Analysis of the synaptic connectivities has revealed that class A and B cholinergic motor neurons make synapses with both the body wall muscle and with class D motor neurons that innervate contralateral body wall muscle (White et al., 1986). Thus, mutations that affect neurotransmission at the cholinergic-GABAergic motor neuron synapse may also lead to a locomotion phenotype. Since the locomotion defect exhibited by the nca-2fgk5); nca-1 (gk9) double mutant may be due to a pre-synaptic defect in the cholinergic motor neuron, it would be interesting to test whether mutations affecting genes in the class D inhibitory motor neurons would enhance or suppress the locomotion defect of the nca-2(gk5); nca-1 (gk9) double mutant. In order to begin addressing this question, different mutants between nca-1, nca-2 and unc-25(el56) were generated. The el56 allele was chosen because it is a predicted loss-of-function allele as the molecular lesion is the introduction of an amber stop codon and it has a severe mutant phenotype (Jin et al., 1999). Figure 36 and Table 14 show that the number of thrashes/min of the nca-2(gk5) unc-25 (el 56); nca-1(gk.9) triple mutant was significantly more (32.9 +/- 4.0; n = 10) (P < 0.05) than that of the nca-2(gk5); nca-l(gk9) double mutant (10.5 +/-1.4; n = 20). This result suggests that a loss-of-function mutation in unc-25 can partially suppress the locomotion defect exhibited by the nca-2(gk5); nca-1 (gk9) double mutant. 205 Figure 36. Thrashing Analysis of unc-25(el56); nca Mutant Strains The thrashing analysis of unc-25 (el 56); nca mutant strains. Bars represent the average of data collected for each strain over a one minute interval and represent mean +/- SEM. Strains marked with an * were significantly different from wild-type (P < 0.05, One-way ANOVA, Origin), but not from one another. The strains marked with a i|/ were significantly different from unc-25 (el 56), unc-25 (el 56); nca-1(gk9) and nca-2 (gk5) unc-25 (el 56) (P < 0.05, One-way ANOVA, Origin). The strain marked with a <|> was significantly different from nca-2(gk5) unc-25(el56); nca-1(gk9) (P < 0.05, One-way ANOVA, Origin). 206 200 H n=10 13 80H M 60-1 H n=20 ^ ^ ^ ^ ^ tSs J}\ ^ ^ < $ r . < r « $ r ^ # ^ 4? 207 Table 14. Summary of Thrashing Rates for Different unc-25(el56); nca Mutant Strains Strain Thrashes/min wild-type 185.9+/-6.8 (n= 10) nca-1 (gk.9) 190.5+/-8.0 (n = 20) nca-2(gk5) 177.8+/-9.4 (n= 10) nca-2 (gk5); nca-1 (gk9) 10.5+/-1.4 (n = 20)* wb unc-25(el56) 107.3+/-3.3 (n= 10)* unc-25(el56); nca-l(gk9) 106.4+/-3.6 (n= 15)* nca-2(gk5) unc-25(el56) 111.3+/-7.6 (n= 10)* nca-2(gk5) unc-25 (el 56); nca-1 (gk9) 32.9+/-4.0 (n=10)*vi/ values are mean +/- SEM * significantly different from wild-type (P < 0.05, One-way ANOVA, Origin) \y significantly different from unc-25(el56) (P < 0.05, One-way ANOVA, Origin) <j» significantly different from nca-2(gk5) unc-25(el56); nca-1(gk9) (P < 0.05, One-way ANOVA, Origin) 208 Analysis of the Defecation Motor Program in nca Mutant Strains Failure Rate of Expulsion Based upon the observations that both the pnca-1 and pnca-2::GFP reporter constructs are expressed in the GABAergic motor neuron DVB (Figures 26,27 and 29) and that the pnca-2::GFP reporter construct is also expressed in cells of the intestine and in the anal depressor muscle (Figure 27), it was hypothesized that mutations in nca-1 or nca-2 may lead to a defect in the Emc step of the defecation motor program and a corresponding increase in the failure rate of expulsion. The Emc step is the last of three sequential muscle contractions of the defecation motor program and is mediated by the neurotransmitter GABA (Mclntire et al., 1993a; Mclntire et al., 1993b). In contrast to the role of GABA as an inhibitory neurotransmitter at the NMJ (Mclntire et al., 1993a; Mclntire et al., 1993b; Bamber et al., 1999; Jin et al., 1999), GABA released from the AVL and DVB motor neurons acts as an excitatory neurotransmitter to cause the simultaneous contraction of the two muscles surrounding the posterior intestine, the anal depressor and anal sphincter muscles, resulting in expulsion of intestinal contents (Beg and Jorgensen, 2003). Figure 37A and Table 15 show that the rates of failure for the single knockout strains nca-1 (gk9) (12.2 +/- 3.2; n = 9) and nca-2(gk5) (10.0 +/- 3.9; n = 10) were not significantly different from wild-type (9.0 +/- 2.8; n = 10) (P > 0.05). In contrast, the failure rate of the double mutant nca-2(gk5); nca-1 (gk9) (27.0 +/- 6.1; n = 10) was significantly different from both wild-type and the two single mutant strains (P < 0.05). These results suggest a possible genetic interaction between nca-1 and nca-2 in the execution of the Emc step of the defecation motor program. 209 Cycle Timing Based upon the observation that the pnca-2::GFP reporter construct is expressed in intestinal cells and that the nca family of VGICs may be activated by intracellular Ca 2 + , it was hypothesized that mutations to nca-2 may lead to a timing defect by uncoupling Ca 2 + signaling with changes to the membrane potential of the intestinal cells (Dal Santo et al., 1999). Figure 37B arid Table 15 show that the timing of the defecation motor program for nca-1 (gk.9) (41.3 +/-1.7; n = 9), nca-2(gk5) (44.1 +/-1.6; n = 10) and nca-2(gk5); nca-1 (gk9) (47.6 +/- 1.6; n = 10) were not significantly different from wild-type (45.4 +/-1.4; n = 10) (P > 0.05). These results suggest that nca-1 and nca-2 probably are not involved in directly regulating the timing of the defecation motor program. 210 Figure 37. Defecation Analysis of the nca Mutant Strains A) The expulsion failure rate of the nca mutant strains are shown as a percentage. Bars represent the pooled values obtained from 9 or 10 animals from each strain observed for 10 consecutive defecation cycles and represent mean +/- SEM. The strain marked with an * was significantly different from wild-type (P < 0.05, One-way ANOVA, Origin). All other strains were not significantly different from one another (P > 0.05, One-way ANOVA, Origin). B) The time between pBocs of the defecation motor program for the nca mutant strains. Bars represent the average of data collected from 9 or 10 animals from each strain observed for 10 consecutive defecation cycles and represent mean +/- SEM. All of the strains were not significantly different from one another (P > 0.05, One-way ANOVA, Origin). 211 A B Table 15. Summary of the Defecation Results for the nca Mutant Strains Strain % Failure Intra pBoc Time (s) wild-type 9.0+/-2.8(n=10) 45.4+/-1.4 (n=10) nca-1 (gk9) 12.2+/-3.2 (n = 9) 41.3+/-1.7 (n = 9) nca-2(gk5) 10.0+/-3.9 (n= 10) 44.1 +/-1.6 (n= 10) nca-2(gk5); nca-l(gk9) 27.0+/-6.1 (n= 10)* 47.6 +/- 1.6 (n= 10) values are mean +/- SEM * significantly different from wild-type (P < 0.05, One-way ANOVA, Origin) 214 Chapter 5 . Discussion The Nematode and Mammalian nca Gene Family The goals of this study were to identify novel four domain-type VGICs and to determine their physiological functions in rat and the nematode model organism, C. elegans. When this project began, a non L-type Ca 2 + channel a i subunit, unc-2, (Schafer and Kenyon, 1995) and an L-type Ca channel a i subunit, egl-19, (Lee et al., 1997) had been previously identified in C. elegans using molecular biology and classical genetic techniques (Figure 18). Screening the C. elegans genome database in early 1997 with oligonucleotide sequences designed against structurally conserved regions present in five different rat HVA Ca channels identified an additional Ca channel a i subunit gene in C. elegans, which we have since named, cca-1 (Figure 18). cca-1 is predicted to encode a T-type Ca 2 + channel and its sequence was subsequently used in our laboratory, as well as in others, to facilitate the molecular cloning of the mammalian T-type Ca 2 + channels (Cribbs et al., 1998; Perez-Reyes et al., 1998; Lee et al., 1999b; McRory etal., 2001). In addition, as a result of the genome-based screening process I have identified two additional four domain-type VGIC a i subunit genes present in the genome of C. elegans. These two genes, named nca-1 and nca-2, appear to be members of a novel family of four domain-type VGICs (Figure 18). The nca-1 and nca-2 sequence information obtained from WormBase was subsequently used in the cloning of full-length cDNAs for nca-1 and nca-2, as well as an nca 215 homologue from rat. Another group used a similar screening strategy to also identify the nca family in C. elegans and ultimately the same rat nca homologue (Rb21) (Lee et al., 1999a). Phylogenetic analysis of gene families present in a range of different organisms has revealed that the mammalian genome has probably experienced two large-scale genome duplications early in chordate evolution (Sidow, 1996). A thorough analysis of the genome of C . elegans supports this model as C. elegans often contains one member of a gene family, whereas mammals routinely contain three or four members (Bargmann, 1998). The results of the screen of the C. elegans genome for novel four domain-type VGICs performed in this study provides some additional support for this model. Figure 18 shows that for each of the three major Ca 2 + channel subfamilies (Non L-type, L-type and T-type) mammals have three or four different cti subunit genes, whereas C . elegans has only a single corresponding ai subunit gene. Interestingly, the number of ai subunit genes present in the genome of C. elegans for the nca family does not follow this pattern of two-fold duplication (Figure 18). Despite repeated attempts to isolate additional nca family members from rat, only one nca family member was isolated in this study. Subsequent examination of the human and rat genome databases as they became available confirmed that there is, in fact, only a single nca gene present in mammals. The nca genes in human (NP 443099) and rat (NP 705894) are located on chromosomes 13 and 15, respectively and have been named VGCNL1 for "voltage-gated channel like 1". Similarly, analysis of the Drosophila genome database revealed the presence of only a single nca gene, called Dmaiu for "unique cti subunit" (or gene product CG1517) located on the X chromosome (Adams et al., 2000; Littleton and Ganetzky, 2000). Interestingly, recent analysis of the working draft of the genome for the closely related nematode, Caenorhabditis briggsae reveals the existence of two nca genes; an nca-1 homologue encoded by gene CBG05508 and an nca-2 homologue encoded by gene CBG18206. Thus, the presence of two nca genes in the genomes of 216 C. elegans and C. briggsae may represent a nematode-specific duplication of the ancestral nca gene and/or may reflect an increase need for the NCA channels in nematode neurobiology. The present genome screen failed to detect any voltage-gated Na + channels in the genome of C. elegans (Figure 18). This finding is consistent with the inability to detect any Na+-based action potentials in electrophysiological recordings made from numerous different neurons in C. elegans (Goodman et al., 1998), as well as in the motor neurons of the related nematode, Ascaris (Davis and Stretton, 1989a, b). The lack of Na + channels in the genome of C. elegans is presumed to be due to a loss of the Na + channel gene(s) during nematode evolution as more "primitive" invertebrates, such as jellyfish have been shown to express functional Na + channels (Anderson, 1987; Anderson et al., 1993). The lack of Na + channel gene(s) in the genome of C. elegans questions the hypothesis that Na + channels are absolutely required in the nervous systems of metazoans to avoid Ca 2 + cytotoxicity under conditions of high neuronal activity (Hille, 1984). This finding also suggests that the nervous system of C. elegans has evolved in such a manner that it no longer requires the physiological properties supplied by Na+ channels and/or the roles performed by the Na+ channels in the nervous systems of other metazoans have been met in C. elegans by channels that can perform analogous functions, perhaps like the nca gene family of four domain-type VGICs. The Genomic Organization of the nca-1 and nca-2 Genes The nca-1 gene spans approximately 14 kb and consists of 28 exons that potentially encode at least two different alternatively spliced proteins of 1797 and 1831 aa (Figure 12). The nca-2 gene spans approximately 14 kb and consists of 23 exons and may undergo alternative splicing to generate at least two different protein products of 1763 and 1785 aa (Figure 14). There are several noteworthy features of the genomic organization of the nca-1 and nca-2 genes. In C. elegans, introns are generally quite short and are frequently smaller than 60 bp, but many 217 genes also contain large introns near their 5' ends. In some instances, it has been determined that the increase in intron size at the 5'end of a gene may be due to the presence of regulatory elements such as alternative promoters and transcriptional enhancers (Blumenthal and Steward, 1997). The introns of nca-1 range in size from 46 bp (intron 4) to 1004 bp (intron 23) with an average length of 312 bp (Figure 12). In contrast, the introns of nca-2 show an overall lower degree of variability in size when compared to nca-1 as the majority of introns in nca-2 range between 48 bp (intron 8) and 463 bp (intron 6). However, introns la and lb of nca-2 are quite large, spanning 4522 bp and 917 bp, respectively (Figure 14). Molecular analysis of the nca-2 gene reveals that there are two different alternative start sites in exon la and lb and the large introns located at the 5' end of nca-2 may be due to the presence of regulatory elements. It is interesting to note that all of the Ca 2 + channel genes in C. elegans (unc-2, egl-19 and cca-1) also contain large introns at their 5'end (C. Thacker and T. Snutch, pers. comm.). There is no obvious relationship between the organization of the intron-exon boundaries of the nca-1 and nca-2 genes and the structural features of the predicted NCA-1 and NCA-2 protein products. For both genes, each domain is encoded by a different number of exons. For example, in the nca-2 gene, domain I is encoded by three exons and each of these exons encode for a different number of transmembrane segments. In contrast, in domain II a similar predicted membrane topology is encoded by six exons. Likewise, domain TTI is encoded by five different exons, whereas domain IV is only encoded by four exons (Figure 14). Furthermore, for both genes, a number of the exons encode portions of adjacent domains. For example, in the nca-1 gene, exon 19 encodes from the end of transmembrane segment S5 of domain LU to the middle of transmembrane segment S4 of domain IV (Figure 12). The voltage-gated Ca 2 + , Na + and NCA channel ct(i) subunits consist of four homologous domains and are thought to have evolved from an ancestral single domain-type voltage-gated ion channel similar to the present day six transmembrane domain K + channels through two rounds of gene duplication (Strong et al., 218 1993). While this may be the case, it is apparent that both the nca-1 and nca-2 genes have diverged substantially from the ancestral four domain-type VGIC and a simple pattern of gene duplication is not readily apparent simply by examining the genomic organization of the two genes. The nca Genes Likely Encode Members of a Novel Family of Voltage-Gated Ion Channels Comparisons Between nca Family Members The longest open reading frames for the nca-1, nca-2 and rat-nca genes encode proteins of 1831,1785 and 1770 aa, respectively. The primary sequences of the NCA proteins are similar to that of other cloned four domain-type VGIC oc(i) subunits and are predicted to consist of four homologous, mainly hydrophobic domains (I-IV), each consisting of six putative membrane-spanning segments (S1-S6) and a pore loop (P-Loop). Comparisons between the predicted protein sequences of the NCA-1 and NCA-2 revealed that they are 57% identical and 68% similar to one another overall with the highest levels of sequence similarity generally occurring in the four transmembrane domains (74% to 88%). Comparison of either NCA-1 or NCA-2 to that of rat-NCA revealed the highest levels of sequence similarity also occurs in the four transmembrane domains (60% to 71%). Interestingly, the highest degree of sequence similarity between the NCA channels is in the domain HI-IV linker (90% for NCA-l/NCA-2 and 78% for NCA-1 or NCA-2/rat-NCA), whereas all other intracellular regions of the NCA channels are far less similar (Figure 17 and Table 4). The high degree of sequence conservation observed for the domain IU-IV linker may be due to the presence of a functionally important structure in this region. For example, the domain nj-IV linker of Na + channels serves as the inactivation gate and is highly conserved between members of the Na + channel family (Catterall, 2000a). Although the amino acid sequence of the domain HI-IV linker of the NCA channels is markedly different from 219 that of Na + channels and does not contain the IFM motif critical for Na + channel inactivation (West et al., 1992), this intracellular loop still may be involved in the inactivation of the NCA channels. Alternatively, the domain III-rV linker of the NCA channels may perform a unique function that endows upon these channels electrophysiological properties that are unique to this family of VGICs. The only sure way of determining the role of the domain IU-TV linker of the NCA channels is to functionally express the NCA channels in a surrogate expression system and determine their electrophysiological properties. Once functional currents have been obtained, the function of the domain III-IV linker could be elucidated through a combination of experiments utilizing chimeric and mutant channels generated by site-directed mutagenesis. Sequence comparisons between the different isoforms of NCA-1 and NCA-2 reveals three major regions of difference. The first major difference is that inclusion of exon la in the NCA-2 channel adds an additional 22 aa to the amino terminal of A5' NCA-2 and this sequence is not present in the NCA-1 channel (Figure 17and Table 4). The second major difference is that inclusion of exon 13 in the NCA-1 channel adds an additional 34 aa to the domain JJ-III linker of NCA-1 AS changing its length from 246 aa to 280 aa and increasing its similarity (from 51% to 56%) to the 277 aa domain II-HI linker of NCA-2 (Figure 17 and Table 4). The other major difference between NCA-1 and NCA-2 is the length of their carboxyl tails; NCA-1 possesses a 341 aa carboxyl tail region, whereas NCA-2 only has 275 aa (Figure 17). Comparisons between the predicted protein sequences of the NCA-1 or NCA-2 proteins and the rat-NCA channel reveals that rat-NCA is 43% identical and 57% similar to both NCA-1 and NCA-2 on an overall basis, consequently no definitive conclusions can be made as to whether rat-NCA is more related to either NCA-1 or NCA-2 when using amino acid similarity as the basis of comparison (Figure 17 and Table 4). The finding that the rat-NCA channel is equally similar to NCA-1 and NCA-2 may lend additional support to the notion that nca-1 and nca-2 are the result of a gene duplication specific to C. elegans and not to a loss of nca genes in the 220 lineages of the other organisms examined. If the latter was the case, then it would be expected that the remaining nca gene present in the genomes of these organisms should show a higher degree of sequence homology to either nca-1 or nca-2, as presumed by their closer evolutionary distance. It is likely that the functional expression of all three channel types in a surrogate expression system such as Xenopus oocytes or mammalian tissue culture cells will be necessary to ascertain the significance of any of the structural differences between the different channels. Comparisons Between the nca Family and Representative Ca2* and Na+ Channels In order to explore the possibility that the nca genes are members of a novel family of four domain-type VGICs, the amino acid sequences of their transmembrane domains were compared with the analogous counterparts of representative Ca 2 + and Na + channels. The amino acid comparisons showed that the Ca 2 + channel family shows higher sequence identity to the Na + channel family (30% identical) than it does to the NCA channel family (20% identical). Similarly, the Na + channel family shows higher sequence identity to the Ca 2 + channel family (30% identical) than it does to the NCA family (20% identical). These findings suggest that on the basis of sequence identities, the NCA channels form a distinct family of four domain-type VGICs separate from that of the previously characterized Ca 2 + and Na + channel families (Figure 18 and Table 5). Cytoplasmic Regions In order to examine possible stracture-function conservation between the nca gene family and the Ca 2 + and Na + channel families, the cytoplasmic regions between the transmembrane domains were examined for any previously characterized protein interaction motifs. Previous work on Ca 2 + channels has revealed that the domain linker of HVA Ca 2 + channel ai subunits contain a consensus sequence (QQ-E--L-GY--WI--E) that is necessary for binding of the 221 accessory (3 subunit to the channel (Pragnell et al., 1994). The domain I-LT linker of the NCA channels lack this consensus sequence and probably do not form a multi-protein complex with Ca 2 + channel (3 subunits. Previous work on mammalian N- and P/Q type Ca 2 + channels has revealed the existence of a "synprint" site in the domain II-ni linker that is required for the physical interaction between the Ca 2 + channel ai subunit and the SNARE proteins syntaxin and SNAP-25 which are involved in the process of vesicle docking and neurotransmitter release (Sheng et al., 1994; Rettig et al., 1996; Sheng et al., 1996; Rettig et al., 1997). The domain H-III linker of the NCA channels lack a readily identifiable "synprint" site and suggests that either the NCA channels are not directly involved in the neurotransmitter release process or that they contribute to release by a mechanism that is different than that characteristized for the N- and P/Q type Ca 2 + channels in vertebrates. This latter notion is supported by the finding that all voltage-gated Ca 2 + channels in C. elegans, Drosophila and other invertebrates lack a "synprint" site (Littleton and Ganetzky, 2000; Spafford et al, 2003; Zamponi, 2003) and indicates that, at least for invertebrates, the existence of a "synprint" site is not a signature for a channel's involvement in the neurotransmitter release process. As mentioned above, previous work on Na + channels has implicated that the domain III-IV linker of Na + channels serves as the inactivation gate for these channels (Vassilev et al., 1988; Stuhmer et al., 1989; Vassilev et al., 1989; Catterall, 2000a) and that the IFM motif located in within this linker is critical to this process (West et al., 1992). The domain III-IV linker of the NCA channels lacks the IFM motif required for Na + channel fast inactivation. S4 Regions While the conserved four domain structures of NCA channels are similar to that of Ca 2 + and Na + channel ct(i) subunits, examination of their S4 regions shows that the putative voltage 222 sensors in the NCA channels are different from that of both Ca 2 + and Na + channels (Figure 19). In general, the S4 regions of the NCA channels contain fewer positively charged residues than either Ca 2 + or Na + channels, especially in domains II and IV. Current models of VGIC activation have assigned the "voltage sensor" role to the membrane-spanning S4 regions of each of the four domains (Armstrong, 1981; Catterall, 1986; Kontis et al., 1997). According to this model, the S4 regions contain positively charged amino acids in every third or fourth position and detect changes in the membrane potential and cause the channel to undergo a conformational change to open the pore (Noda et al., 1984; Tanabe etal., 1987; Ellis et al., 1988). Experiments have shown that the conformational change induced by the S4 regions are due to the physical movement of these segments through the transmembrane pathway formed by the rest of the channel (Yang and Horn, 1995; Aggarwal and MacKinnon, 1996). Since the S4 regions contain regularly spaced positively charged residues, movement of the S4 segments through the membrane also results in the movement of charged amino acids. This movement of positively charged amino acids produces a small, but detectable current called the gating current and the size of this gating current is determined by the number of charges that move in response to a change in membrane potential (Armstrong, 1981). It has been hypothesized that the actual number of charges that move through the membrane is what determines the voltage dependence of activation for a given type of ion channel (Yellen, 1998). In vitro site-directed mutagenesis experiments on Na + and K + channels have found that neutralizing the positively charged amino acids in the S4 regions produces changes to the activation properties of VGICs. These experiments have shown that individual charge neutralizing substitutions can produce positive shifts in the voltage-dependence of activation, reduce the steepness of the activation curve and can result in smaller gating currents (Stuhmer et al., 1989; Papazian et al, 1991; Aggarwal and MacKinnon, 1996; Seoh et al., 1996). Furthermore, experiments with Na + channels have found that the positively charged residues 223 within S4 regions contribute unequally to the voltage-dependent properties and that each S4 region as a whole makes unequal contributions to this process (Stuhmer et al., 1989; Kontis et al., 1997). Overall, the aforementioned changes to the voltage dependence of activation reflect a decrease in sensitivity of mutant channels to changes in membrane potential. Since decreasing the total amount of charge in the S4 region seems to decrease voltage sensitivity, it is hypothesized that the fewer number of positively charged residues present in the S4 regions of the NCA channels (Figure 19) may reflect a need for strong membrane depolarization to activate these channels. In addition, the fewer number of positive charges in the S4 regions of the NCA channels may endow on these channels other unique activation properties. Models for the inactivation process of some VGICs such as Na + channels hypothesize that most the voltage dependence of the inactivation process is coupled to that of activation (Armstrong, 1981). In vitro site-directed mutagenesis experiments on Na + channels have shown that amino acid substitutions of the positively charged residues in the S4 regions also have different effects on the voltage-dependent properties of inactivation (Chen et al., 1996; Kontis and Goldin, 1997). These experiments have shown that individual amino acid changes can reduce the slope of the steady-state inactivation curve and decrease the rate of inactivation in a voltage-dependent manner with the most profound effects seen when the changes are made to the S4 region of domain IV (Chen et al., 1996; Kontis and Goldin, 1997). Since the domain IV S4 region of the NCA channels contain fewer positively charged residues than either Ca 2 + or Na + channels it is possible that the NCA channels may inactivate relatively slowly and display other unique inactivation properties. P-Loop Regions The involvement of specific amino acids within the P-Loop regions in ion selectivity was first demonstrated by in vitro site-directed mutagenesis experiments on voltage-gated Na + 224 channels. In these experiments its was found that for Na + channels (DEKA) only two amino acid substitutions K1422E and A1714E in the domain III and domain IV P-Loops, respectively were necessary to change ion selectivity (DEKA to DEEE) from monovalent (Na*) selective to divalent (Ca2+) selective (Heinemann et al., 1992). In addition, these experiments also found that the single amino acid substitution A1714E in the domain IV P-Loop by itself changed the ability of the Na + channel (DEKA to DEKE) to properly discriminate between monovalent cations (Na+ versus K*) (Heinemann et al., 1992). These results involving the ion selectivity motif in Na + channels implied that the corresponding amino acid residues in the four P-Loop regions of Ca 2 + channels (EEEE or EEDD) may also be important in determining ion selectivity for the Ca 2 + channels. This hypothesis was confirmed by in vitro site-directed mutagenesis experiments which showed that a ring of negative charges formed by glutamate residues (E) from the P-Loop of each of the four domains is critical in determining the ion selectivity of L-type HVA Ca 2 + channels (Kim et al., 1993; Tang et al., 1993; Yang et al, 1993). Mutations of any of these glutamate (E) residues dramatically decreased the selective permeation of divalent over monovalent cations (Kim et al., 1993; Yang et al., 1993; Yatani et al, 1994; Ellinor et al., 1995; Parent and Gopalakrishnan, 1995). According to proposed models of Ca 2 + channel ion selectivity, it appears that the high affinity binding site(s) responsible for Ca 2 + selectivity are formed by a ring of negatively charged amino acids present in the pore of Ca 2 + channels and that each negative residue contributes differently to Ca 2 + affinity, selectivity and permeation. For example, amino acid substitution of the domain III glutamate has the greatest effect on ion selectivity and permeation, as making the amino acid substitution E1086K in the cardiac Ca 2 + channel (EEEE to EEKE) changed the ion selectivity from divalent to monovalent selective (Tang et al., 1993; Yang et al., 1993; Ellinor et al., 1995). These studies involving both Na + and Ca 2 + channels also showed that it is the presence 225 of a lysine (K) in the domain HI P-Loop that determines monovalent selectivity (Heinemann et al., 1992; Tang et al., 1993; Yang et al., 1993; Ellinor et al., 1995). While the conserved four domain structures of the NCA channels are similar to that of Ca and Na channel ct(i) subunits, examination of their pore regions shows that the ion selectivity motif in the NCA channels (EEKE) is different from that of both Ca 2 + (EEEE or EEDD) and Na + channels (DEKA) (Figure 20). Unlike Ca 2 + channels but like Na + channels, the NCA channels contain a lysine (K) in the P-Loop of domain III. Furthermore, similar to HVA Ca channels, but unlike that for Na channels, the NCA channels contain a glutamate (E) in the P-Loop of domain IV. The presence of a lysine (K) residue in the domain HI P-Loop of the NCA channels (EEKE) suggests that these channels may preferentially permeate monovalent cations, as a lysine (K) in this position is critical in monovalent versus divalent selectivity (Heinemann et al, 1992; Tang et al., 1993; Yang et al., 1993; Ellinor et al., 1995). It should be noted however that the actual effectiveness of the lysine (K) residue in the domain IH P-Loop of NCA channels in excluding divalent cations, such as Ca 2 + , from permeating the channel still remains to be determined. In addition, the presence of a glutamate (E) in the domain IV P-Loop of the NCA channels (EEKE) suggests that they may have a decreased ability to discriminate amongst various monovalent cations. This assumption is based upon the finding that when the alanine (A) in the domain IV P-Loop was changed to a glutamate (E) in a Na + channel (DEKA to DEKE) the corresponding mutant channel was no longer able to properly select between monovalent cations (Heinemann et al., 1992). Thus, based upon these in vitro mutagenesis experiments it is hypothesized that the NCA channels might represent a novel family of voltage-gated, non-selective cation channels. 226 NCA Channel Expression and Electrophysiological Analysis In order to test the aforementioned hypotheses concerning the ion selectivity and electrophysiological properties of the nca family of four domain-type VGICs, the full-length rat-nca A and nca-2 cDNAs were transiently transfected into HEK cells and currents were assayed using the patch clamp technique. Despite repeated attempts, no functional currents were obtained for the rat-NCA A and the NCA-2 channels transiently transfected into the HEK tsA201 tissue culture cell line. There may be a number of reasons as to why no inward currents attributed to the NCA channels were obtained. Previous work on VGICs has shown that transient expression of newly isolated ion channels into surrogate expression systems has not always resulted in detectable currents. Examples include, the atypical Na + channel, Na x (George et al., 1992) and a jellyfish four domain-type Na + channel (Anderson et al., 1993). One possible explanation for the lack of functional expression of these channels is that they require coexpression of additional accessory subunits for the proper folding and assembly into functional channels, analogous to the roles provided by the accessory subunits of the HVA Ca 2 + (Bichet et al., 2000) and Na + (Schmidt et al., 1985) channels. In order to address this possibility, the rat-NCA A channel was separately coexpressed with both the Ca 2 + channel 0128-1 and pn> (or p2a) accessory subunits or with the Na + channel Pi accessory subunit. Unfortunately, none of these coexpression experiments resulted in detectable whole cell currents that could be attributed to the rat-NCA A channel. The lack of functional rat-NCA A currents when the channel was coexpressed with either the Ca 2 + or Na + channel accessory subunits raises the possibility that the NCA channels may have their own unique set of accessory subunits that may be structurally different from those already identified for HVA Ca 2 + and Na + channels. If this is the case, then putative NCA channel accessory subunits may not be readily identifiable by screening genome databases using homology based searches. Consequently, a combination of molecular genetic and biochemical 227 approaches may be necessary to determine the subunit composition of the NCA channel complex and the identity of their accessory subunit(s). Another possible explanation for the lack of functional currents obtained for the rat-NCA A and NCA-2 channels is that activation of the NCA family of VGICs may require an as yet to be defined combination of conditions to initiate channel opening. For example, activation of the NCA channels may require a particular second messenger activation in addition to membrane depolarization. Early electrophysiological recordings from cultured cardiac cells revealed the presence of non-selective cation channels that are activated by intracellular Ca 2 + (Colquhoun et al., 1981). Since that time, numerous other non-selective cation channels have been identified that are activated by intracellular Ca 2 +. This presumably heterogeneous class of ion channels have been grouped together based upon there common requirement of Ca 2 + for activation and have collectively been termed Ca2+-activated non-selective cation (CAN) channels (Petersen, 2002). Despite their analysis in native cells, the molecular identity of the CAN channels still remains to be determined (Petersen, 2002). One such type of CAN channel expressed in rat hippocampal neurons was found to be activated by nVmediated Ca 2 + release following activation of the metabotropic glutamate receptor, type-1 (mGluRl) and resulted in prolongation of membrane depolarization (Partridge and Valenzuela, 1999). Perhaps the NCA channels are similar to this type of CAN channel requiring intracellular Ca 2 + for activation and may also serve a similar role in neurophysiological processes. Experiments on a different type of CAN channel expressed in mouse hippocampal neurons revealed that decreasing the concentration of extracellular Ca 2 + activated the CAN channel, whereas decreasing the pH of the external recording solution to 6.5 inhibited the channel. In contrast, increasing the external pH to 8.5 potentiated the current through this CAN channel (Chu et al., 2003). Thus, some CAN channels may be sensitive to both Ca 2 + and pH. It is possible that the particular recording conditions used in each of the two NCA channel 228 experimental protocols were inappropriate for detecting NCA currents if these channels have similar sensitivities to Ca 2 + and/or pH as the aforementioned CAN channels. A number of different ionic conditions and voltage protocols may have to be tested in order to obtain functional NCA currents. Another possible explanation for the lack of functional rat-NCA A currents is that the two amino acid changes in the rat-NCA A channel, S52P in the domain I SI segment and A748T in the domain II-III linker, may affect expression in HEK cells. These putative cloning artifact errors in the rat-nca A cDNA need to be corrected and the newly altered clone needs to be retested for functional expression. It should be noted however that attempts to express a different rat-nca cDNA, Rb21, that does not contain the S52P and A748T amino acid changes also failed to generate functional currents when expressed in either Xenopus oocytes or HEK tsA201 cells (Lee et al, 1999a). Similarly, an additional independently isolated rat-nca cDNA also failed to yield functional currents when expressed in surrogate systems (M. Tang and L. Kaczmarek, pers. comm.). The fact that three independently isolated rat-nca cDNAs from separate laboratories all failed to generate functional currents when expressed in different surrogate systems suggests that the nca family of VGICs may not be easily studied using standard approaches. In the meanwhile, a genetically amenable model system such as C. elegans may prove useful at providing information with regards to the biological roles and physiological functions of the nca family of four domain-type VGICs. Deletion Mutations in the nca-1 and nca-2 Genes The nca-1 and nca-2 genes were first identified by screening the C. elegans genome data base and no visible mutations had previously been reported for either of these two genes. The inability of forward genetics to generate any visible mutations in either nca-1 or nca-2 exemplifies a potential limitation of forward genetics in identifying new genes of interest, 229 especially for members of multigene families. The availability of the genomic sequence of nca-1 and nca-2 made it possible to generate targeted putative loss-of-function mutations in these two genes. Examination of the molecular lesions in nca-1 (gk.9) and nca-2(gk5) revealed large deletions in these two alleles that removed almost entire domains from each of the respective protein products and changed their subsequent reading frames (Figure 25). The 5' breakpoint of the deletion mutation in nca-1(gk9) resides within the domain JJ-II linker (Figure 25A) and might allow for the expression of a two domain-type truncated product that could form a functional VGIC by dimerization. Although theoretically possible, coexpression of a truncated construct of the Ca channel Cav2.2 representing domains I and II did not form functional channels when expressed individually or together with the accessory subunits Pib and cc2S-l in COS-7 cells (Raghib et al., 2001). Thus, based upon the size of the deletions and the fact the both these mutations would lead to prematurely truncated proteins, both of the nca-1 (gk9) and nca-2(gk5) mutations are predicted to reflect hull phenotypes. The nca-1(gk.9) and nca-2(gk5) single knockout strains were examined for visible phenotypes, such as defects in locomotion, as well as for morphological abnormalities. Visibly, both of the single mutant strains exhibited wild-type patterns of locomotion and their overall size appeared to be normal. Furthermore, there were no gross physical abnormalities in their shape and body construction. Taken together, these observations suggest that neither nca-1 and nca-2 alone are critically involved in producing a wild-type body plan or in generating wild-type movement. In contrast, mutations to the nca homologue in Drosophila, Dmotiu, cause subtle changes to the morphology of the fly, such as a physical narrowing of the abdomen and an overall slight decrease in body size (Krishnan and Nash, 1990; Nash et al, 2002). Furthermore, flies with mutations in the nca homologue exhibit a "hesitant walking" phenotype which is characterized by a tendency for the flies to walk in an atypical manner consisting of walking for a few steps, stopping and then walking again (Krishnan and Nash, 1990). The phenotypes 230 displayed by these mutant flies suggest that the nca homologue in Drosophila is involved in both determining body morphology and in the generation of movement. These observed differences between the nca-1(gk9) and nca-2(gk5) single mutant strains in C. elegans and the nca mutants in Drosophila may represent species-specific differences in NCA channel biology and/or tissue expression, or they may reflect the possibility of functional redundancy between nca-1 and nca-2 in C. elegans (see below; NCA-1 and NCA-2 are Involved in Acetylcholine Release at the Neuromuscular Junction). The Expression Pattern of the nca Family of Voltage-Gated Ion Channels The lack of any readily noticeable mutant phenotype for either the nca-1 (gk.9) and nca-2(gk5) single mutant strains was somewhat surprising considering that mutations in the Drosophila nca homologue cause a number of observable phenotypes including a narrower abdomen, "hesitant walking", lower brood sizes and an altered sensitivity to the anesthetic halothane (Krishnan and Nash, 1990; Campbell and Nash, 1994; Nash et al., 2002). One possible explanation for the lack of any observable phenotypes for the nca-1 (gk9) and nca-2(gk5) single mutant strains is that there may be functional interactions or genetic redundancy between the two nca genes in C. elegans. In order to explore this possibility the cellular expression patterns of the nca-1 and nca-2 genes were determined using promoter: :GFP fusion constructs. Examination of the transgenic strain TS48 vals6 that expressed the integrated p«ca-7::GFP reporter construct showed GFP expression primarily in the nervous system (Figure 26). Of particular interest was the reporter construct's expression in numerous nerve ring interneurons, in the class A, B and AS cholinergic motor neurons of the dorsal and ventral nerve cords (Figures 26B and 28) and in the GABAergic neuron DVB (Figures 26C and 29). Similarly, examination of the transgenic strain TS191 vals!5 that expressed the integrated pnca-2:.GFP reporter construct also showed GFP expression in the 231 nervous system (Figure 27). Interestingly, the pnca-2::GFP reporter construct was also found to be expressed in the same class A, B and AS cholinergic motor neurons that also expressed the pnac-7::GFP reporter construct (Figures 27B and 28). Coexpression of the two different reporter constructs in the same neurons within the dorsal and ventral nerve cords lends additional support to the hypothesis that the nca-1 and nca-2 genes may perform functionally redundant roles within the cholinergic motor neurons (Figure 28). The pnca-2::GFP reporter construct was also found to be expressed in the class D GABAergic motor neurons of the dorsal and ventral nerve cords (Figures 27B and 28), in the GABAergic neuron DVB (Figures 27C and 29), as well as in other non-neuronal tissues (Figure 27). The broader cellular expression pattern of the pnca-2::GFP reporter construct suggests that the NCA-2 channel may have a more widespread role in the cellular physiology of the worm as a whole than the NCA-1 channel. The observed expression of the pnca-1 and pnca-2::GFP reporter constructs in both cholinergic and GABAergic neurons in C. elegans is similar to that reported for the distribution of the nca homologue in Drosophila where immunohistochemistry experiments have revealed that adult fly heads display a staining pattern that is similar to that reported for markers of cholinergic and GABAergic neurons (Nash et al., 2002). Furthermore, a survey of fly tissues taken at various developmental stages showed that the adult head is the richest source of the Dmaiu channel protein in Drosophila suggesting that nca gene in flies likely plays a more prominent role in neurophysiology than in other tissue systems (Nash et al., 2002). Overall, the finding that Dmctiu is expressed at its highest levels in the CNS of Drosophila is similar to that in C. elegans, as both the pnca-1 and pnca-2::GFP reporter constructs were mainly expressed in the nervous system of the worm (Figures 26 and 27). In contrast to the primarily neuronal expression of the nca gene(s) in both C. elegans and Drosophila, nca gene expression in rat seems to be more widespread as rat-nca RNA was detected in various different brain regions, as well as in a variety of organ tissues (Figure 21). 232 This apparently more widespread distribution of the nca gene in rat may reflect a species-specific diversification of the role of the NCA ion channel in mammals or may reflect an artifact of the detection strategies used to assay NCA channel expression in the three different organisms. For example, the use of RT-PCR in combination with Southern blot analysis to detect the nca gene expression pattern in rat may be more sensitive at detecting lower levels of channel expression than the immunohistochemical technique used to detect nca gene expression in Drosophila. Furthermore, the immunohistochemistry experiment that examined the tissue expression profile of the nca gene in Drosophila only qualitatively stated that the highest level of Dmcxiu expression was found in adult fly heads and never mentioned whether staining was observed in any other tissues (Nash et al., 2002). Of note however, the narrow abdomen phenotype observed in strains with mutations in the Drosophila nca gene suggests the possibility of a more widespread expression pattern of the Dmctiu channel and additional biological roles. Similarly, the difference in the expression pattern observed for the nca gene in rat as compared to that of nca-1 and nca-2 may be due to the nature of the reporter constructs used to examine the expression of these two genes in C. elegans. The promoter: :GFP reporter constructs used to examine the expression of the nca-1 and nca-2 genes may not have contained all of the necessary promoter and regulatory elements needed to exactly recapitulate the wild-type expression pattern of the nca-1 and/or nca-2 genes. This notion is supported by the observation that the transgenic strains TS48 vals6 and TS191 valsl5 that expressed the integrated pnca-1 and pnca-2::GFP reporter constructs, respectively exhibited some mosaic GFP expression. Thus, the observed expression profiles for the two GFP reporter constructs used in this study may not reflect the entire or correct expression patterns of these two genes. In order to rule out this possibility a combination of in situ hybridization and immunohistochemical techniques could be used to confirm the observed expression pattern of the nca-1 and nca-2 genes. Interestingly, although the pnca-2::GFP reporter construct was primarily expressed in the nervous system in C. 233 elegans, GFP expression was also observed in a number of non-neuronal tissues in the worm (Figure 27). The expression of the pnca-2: :GFP reporter construct in non-neuronal tissues suggests that the nca genes as a whole in C. elegans may have a more widespread expression pattern than that reported for Drosophila, but possibly not as broad as that observed in mammals. Examination of the expression pattern of the nca genes in C. elegans and rat also revealed another interesting observation. In this study, RT-PCR in combination with Southern blot analysis was used to detect rat-nca RNA expression in various tissues and revealed rat-nca gene expression in the seminal vesicles and ovaries of male and female rats, respectively (Figure 21). Similarly, the pnca-2: :GFP reporter construct was observed to be expressed in the vulval muscles in the worm (Figure 27) and in the distal tip cells of the gonad (data not shown). The expression of NCA channel proteins in tissues involved in reproduction in both rat and C. elegans is consistent with the observation that flies with mutations in the Drosophila nca gene, Dmctiu, have lower brood sizes as compared to wild-type (Krishnan and Nash, 1990). Although egg-laying defects and brood size were not measured for the C. elegans nca mutant strains in this study, the aforementioned observations may suggest a role for the NCA channels in reproductive biology and is an interesting area of future research. NCA-1 and NCA-2 are Involved in Acetylcholine Release at the Neuromuscular Junction Research in C. elegans neurobiology has progressed to the stage where biological functions have been assigned to a number of specific neurons and behavioral assays have been developed to exploit functional deficits within various neural circuits. By determining both the cellular expression pattern of the gene of interest and the biological functions of those cells, the likelihood of finding a behavioral phenotype and insight into the biological roles and physiological functions of this gene is greatly increased. 234 Based upon the observation that pnca-1 and pnca-2.:GFP reporter constructs were both expressed in the cholinergic motor neurons of the dorsal and ventral nerve cords (Figures 26,27 and 28), it was hypothesized that the lack of a locomotion phenotype for either of the nca-1 (gk9) and nca-2(gk5) single knockout strains may be due to functional redundancy between these two genes. In order to address this question, the nca-2(gk5); nca-1 (gk9) double mutant was generated. Examination of the nca-2 (gk5); nca-1 (gk9) double mutant on seeded agar plates revealed a locomotion phenotype that consisted of the animal cycling between periods of inactivity and episodes of short forward or backward movement. This locomotion phenotype exhibited by the nca-2(gk5); nca-1 (gk9) double mutant strain is remarkably similar to the "hesitant walking" phenotype reported for the Drosophila nca mutants (Krishnan and Nash, 1990) and may suggest a common role for the NCA channels in generating locomotion. Since both the pnca-1 and p«ea-2::GFP reporter constructs were expressed in the cholinergic motor neurons of the dorsal and ventral nerve cords, it was hypothesized that the locomotion defect observed for the nca-2(gk5); nca-1(gk9) double mutant strain could be attributed to alterations in cholinergic neurotransmission at the NMJ. The results of the biochemical assays showed that the nca-2(gk5); nca-1 (gk.9) double mutant was mildly, but significantly resistant to aldicarb (Figure 31 and Table 9) and showed wild-type sensitivity to nicotine (Table 9). These findings are consistent with mutations in genes that are directly involved in neurotransmitter release at the cholinergic NMJ, such as unc-2. The non L-type Ca 2 + channel, UNC-2, functions as the major Ca 2 + channel involved in triggering neurotransmitter release at the NMJ. unc-2 mutant animals are resistant to aldicarb, but sensitive to levamisole and nicotine, likely because there is a significant decrease in the amount of ACh released at the NMJ (Miller et al, 1996; Richmond et al, 2001; Mathews et al., 2003). Taken together, the results suggest that NCA-1 and NCA-2 contribute pre-synaptically to cholinergic neurotransmission at the NMJ. 235 Examination of the nca-2(gk5); nca-1 (gk.9) double mutant strain using the body bends and thrashing assays revealed that the worm's ability to properly generate movement was dramatically affected by loss of both NCA-1 and NCA-2 as the locomotion phenotype of the nca-2(gk5); nca-1 (gk.9) double mutant strain was quite severe when compared to wild-type (Figure 30 and Table 8). Other than the locomotion phenotype, the nca-2(gk5); nca-1 (gk9) double mutant strain did not show any obvious morphological abnormalities that is in contrast to Drosophila nca homologue mutants. This lack of any obvious changes to the body plan of the double nca mutant in C. elegans suggests that the role of the nca gene in Drosophila body architecture may be fly-specific. A Hypothetical Model for the Role of NCA-1 and NCA-2 in Cholinergic Motor Neurons The lack of identifiable Na + channels in the C. elegans genome combined with the inability to detect any Na + channel activity in electrophysiological recordings of neurons (Goodman et al., 1998) has led to the hypothesis that electrical signals in the neurons of C. elegans are not propagated by action potentials, but instead are transmitted by electrotonic spread (Davis and Stretton, 1989a, b; Goodman et al., 1998). Since neurons in C. elegans are relatively small, have high membrane resistance and are nearly isopotential, this passive mode of electrical signaling is possible without the large-scale amplification normally provided by Na + channels (Davis and Stretton, 1989a, b; Goodman et al., 1998). In contrast to that observed in neurons, electrical signaling in the pharyngeal and body wall muscles of C. elegans is dependent on the generation of Ca2+-based action potentials mediated by the EGL-19 putative L-type Ca 2 + channel (Lee et al., 1997; Jospin et al., 2002). Although the subcellular localization of the NCA-1 and NCA-2 ion channels was not determined in this study, immunohistochemistry experiments in Drosophila have revealed that the fly nca homologue, Dmotiu, is localized within regions of high synaptic density in the adult 236 fly brain and not in regions occupied by cell bodies or axon projections (Nash et al., 2002). This observation suggests that the nca family of ion channels in Drosophila, and perhaps in C. elegans, are either involved in the process of neurotransmitter release from pre-synaptic terminals or in the modulation of neurotransmitter affects in the post-synaptic cell. A hypothetical model that may help explain the roles of nca-1 and nca-2 in cholinergic neurotransmission at the NMJ proposes that NCA-1 and NCA-2 are non-selective cation channels that function redundantly to ensure the effective transmission of electrical signals in cholinergic motor neurons from the command interneuron-motor neuron synapse to the NMJ by providing an additional source of membrane depolarization. This additional source of depolarization provided by both NCA-1 and NCA-2 would conceivably facilitate electrical conduction in neurons relying solely on the passive mode of electrical signaling. Thus, the loss of both the NCA-1 and NCA-2 ion channels in cholinergic motor neurons impairs their ability to properly propagate the electrical signals received from the command interneurons. This would ultimately lead to a reduction in the amount of ACh released at the NMJ and a corresponding movement defect as not all electrical signals would be propagated faithfully to the nerve terminals (Figure 38). According to the model, if loss of both the NCA-1 and NCA-2 ion channels in cholinergic motor neurons alters the neurons ability to propagate electrical signals to the axon terminals, then mutations in both nca-1 and nca-2 may enhance additional downstream mutations that directly affect neurotransmitter release and enhance their locomotion deficits. Some examples of these downstream genes that directly affect neurotransmitter release when mutated include; unc-2 (a non L-type Ca 2 + channel) (Schafer and Kenyon, 1995; Mathews et al., 2003), snt-1 (synaptotagmin) (Nonet et al., 1993), snb-1 (synaptobrevin) (Nonet et al., 1998), unc-64 (syntaxin) (Saifee et al., 1998) and ric-4 (SNAP-25) (Nguyen et al., 1995; Miller et al, 1996). Consistent with this model, the nca-2(gk5); nca-1 (gk9) double mutant was found to 237 Figure 38. A Hypothetical Model for the Role of NCA-1 and NCA-2 in Cholinergic Motor Neurons According to this hypothetical model, NCA-1 (blue oval) and NCA-2 (green oval) are non-selective cation channels that function redundantly to ensure the effective transmission of graded electrical signals (red arrow) within cholinergic motor neurons. Therefore, loss of both the NCA-1 and NCA-2 ion channels impairs the ability of the motor neuron to properly propagate the electrical signals received from the command interneurons (CIN). This would ultimately lead to a reduction in the amount of ACh released at the NMJ and a corresponding movement defect as not all electrical signals would be propagated faithfully to the nerve terminals and cause body wall muscle (BWM) contraction through the activation of the nicotinic ACh receptors (nAChR, purple rectangles). According to this model, loss of both the NCA-1 and NCA-2 should enhance additional downstream defects caused by exposure to pharmacological agents, such as aldicarb or halothane, or by mutations that cause defects in neurotransmitter release. Examples of the latter include mutations to the UNC-2 Ca 2 + channel (yellow oval) which is directly involved in the release of neurotransmitters at the NMJ and perhaps other Ca 2 + channels (yellow ovals) that may be involved in the release process (EGL-19). 238 239 enhance the effect of two different unc-2 mutations (see below; Mutations to both nca-1 and nca-2 Enhance the Locomotion Defects of unc-2 Mutants). The Effect of Halothane on the nca-2(gkS); nca-l(gk9) Double Mutant Strain The hypothetical model for the role of the NCA-1 and NCA-2 ion channels in signal propagation (Figure 38) is supported by the results of our halothane assay where it was found that the nca-2(gk5); nca-1(gk9) double mutant was significantly hypersensitive to the anesthetic (Figure 32 and Table 9). If it is assumed that the locomotion defect exhibited by the nca-2(gk5); nca-1 (gk.9) double mutant is due to an upstream signaling defect, then exposure to halothane which depresses pre-synaptic neurotransmission at the NMJ (Nishikawa and Kidokoro, 1999) should exacerbate the movement deficit. The hypersensitivity of the nca-2(gk5); nca-1(gk9) double mutant to halothane appears to be different from that originally reported for mutations in the nca homologue in Drosophila, Dmaiu. The Dmaiu gene (originally called na for narrow abdomen) was originally identified in a screen for halothane resistant mutants (Krishnan and Nash, 1990). In this screen the authors used an inebriometer consisting of a glass column containing a number of nylon mesh baffles at different heights to screen for halothane resistant mutants (Krishnan and Nash, 1990). When exposed to halothane, wild-type adult flies tend to fall off of the baffle that they are on and eventually settle on a lower one. After prolonged exposure to halothane, wild-type flies go through a series of falls landing on lower and lower baffles until they are finally eluted off of the column. According to this assay, flies that elute off of the column at significantly later times than that of wild-type flies are considered to be halothane resistant (Krishnan and Nash, 1990). Two mutant strains of the Dmaiu (or na) gene, har38 and har85, were originally considered to be halothane resistant because almost all of the mutant flies for each of these two strains stayed in the inebriometer. However, upon closer inspection of the har38 and har85 mutant strains, it was 240 noticed that these mutant flies actually became paralyzed and "frozen" upright at lower concentrations of halothane than wild-type flies. This observation suggested that the har38 and har85 mutant strains are actually hypersensitive to halothane, but failed to elute off of the column due to the paralysis induced by exposure to halothane (Krishnan and Nash, 1990). The putative hypersensitivity of the har38 and har85 mutant strains to halothane was later reconfirmed in an independent assay where the response of adult flies to a noxious light stimulus was measured and found that the har38 and har85 mutant strains displayed increased movement in response to this noxious stimulus at lower halothane concentrations as compared to wild-type flies indicating a hypersensitive response (Campbell and Nash, 1994). While its mechanism of action is not well understood, previous work on the effects of halothane on glutamate-mediated synaptic transmission at the NMJ in Drosophila larvae has shown that in the presence of halothane there is a marked reduction in the amplitude of nerve-evoked excitatory junctional currents (EJCs) in wild-type larvae. This result suggests that at least one of the halothane-mediated effects is to depress pre-synaptic neurotransmission at the NMJ in Drosophila (Nishikawa and Kidokoro, 1999). In contrast to that observed for wild-type flies, there was no reduction in the amplitude of nerve-evoked EJCs for the har38 and har85 mutant strains in the presence of halothane (Nishikawa and Kidokoro, 1999). One possible explanation for this discrepancy is that the halothane assays were performed on adult flies (Krishnan and Nash, 1990; Campbell and Nash, 1994), whereas the NMJ recordings were performed on larvae (Nishikawa and Kidokoro, 1999) and perhaps there may be a developmental component to the halothane-mediated effects observed in Drosophila. Another possible explanation is that the hypersensitivity to halothane exhibited by the har38 and har85 mutant strains may not be due to an additional reduction in the amount of glutamate released at the NMJ in Drosophila, but rather to another site of action, perhaps in the ellipsoid body in the CNS where the Dmaiu channel is also highly expressed (Nash et al., 2002). 241 Although the Drosophila NMJ recordings in the presence of halothane of har mutants are in contrast to that proposed by the aforementioned model, previous studies in C. elegans have found that the predominant mechanism for the effect of halothane on the NMJ and locomotion appears to be a pre-synaptic mechanism (van Swinderen et al., 1999). These studies have shown that mutations in genes encoding synaptic transmitter release proteins such as syntaxin and SNAP-25 have altered sensitivity to halothane due to presumed changes in the amount of cholinergic neurotransmission at the NMJ (van Swinderen et al., 1999; van Swinderen et al., 2001). The observed differences in the mechanism of halothane action at the NMJ in Drosophila and C. elegans nca mutants may reflect differences in the expression pattern of their respective NCA channels or alternatively to neurophysiological differences between the glutamatergic motor neurons of Drosophila and the cholinergic motor neurons of C. elegans. In order to confirm the proposed model for the effects of halothane on C. elegans on pre-synaptic cholinergic neurotransmission, electrophysiological recordings at the NMJ (Richmond et al., 1999; Richmond and Jorgensen, 1999) should be performed for both wild-type and the nca-2(gk5); nca-1 (gk9) double mutant strain in the presence and absence of halothane. Mutations to both nca-1 and nca-2 Enhance the Locomotion Defects oi unc-2 Mutants If loss of both NCA-1 and NCA-2 in cholinergic motor neurons alter their ability to propagate electrical signals to axon terminals, then loss-of function mutations in both nca-1 and nca-2 would be predicted to enhance mutations in other genes that contribute directly to neurotransmitter release. Behavioral, pharmacological and electrophysiological studies have suggested that the non L-type Ca 2 + channel, UNC-2, functions as the major Ca 2 + channel involved in triggering neurotransmitter release at the NMJ (Miller et al., 1996; Richmond et al., 2001; Mathews et al, 2003). Animals with reduction-of-function or loss-of-function mutations in unc-2 display a range of behavioral abnormalities characteristic of neurotransmission defects 242 including slow, kinked movement and difficulty with backing (Brenner, 1974; Schafer and Kenyon, 1995; Rand and Nonet, 1997). unc-2 mutant animals are resistant to aldicarb, but sensitive to levamisole and nicotine, implicating UNC-2 in cholinergic neurotransmission at the NMJ (Miller et al., 1996; Mathews et al., 2003). In addition, electrophysiological recordings from loss-of-function unc-2(e55) mutant animals show an ~3 fold decrease in evoked release at the NMJ further implicating UNC-2 in the process of neurotransmitter release (Richmond et al., 2001). Furthermore, the severity of the movement deficit exhibited by animals with mutations in the unc-2 gene depends on the type of genetic mutation. Animals with presumed reduction-of-function mutations to unc-2 such as ox8 show moderate to mild defects in movement, whereas animals with presumed loss-of-function mutations to unc-2 such as el29 show severe defects in movement (Mathews et al., 2003). Differences in the severity of locomotion defects for the different classes of mutations in unc-2 are predicted to be correlated with the amount of Ca entry through mutant UNC-2 channels and the corresponding amount of subsequent neurotransmitter released (Mathews et al., 2003). To address the hypothesis that loss of both NCA-1 and NCA-2 in cholinergic motor neurons may enhance downstream defects in neurotransmitter release, various double and triple mutants between different unc-2 alleles and nca-1(gk9) and nca-2(gk5) were generated and the severity of their locomotion defects were quantified using the thrashing assay. It was assumed that significant decreases in the amount of ACh released at the NMJ would result in an ' enhancement of the locomotion defect and could be quantified using the thrashing assay. Furthermore, the thrashing assay was used as a means of assessing the overall ability of the different mutant strains to move because animals display non-directional thrashing behavior in liquid (Croll, 1975) as opposed to directional movement on seeded agar plates. In this regard, the number of thrashes per minute in liquid should not be influenced by changes in direction and should only reflect the ability to move as a whole. The aldicarb assay could not be used to 243 quantify any additional defect in ACh release at the NMJ as all of the unc-2 strains used were 100% resistant to aldicarb over the two hour period examined (data not shown). One of the unc-2 alleles used was the putative reduction-of function 0x8 allele which is thought to alter the voltage-dependent properties of the UNC-2 Ca 2 + channel and decrease the amount of pre-synaptic Ca 2 + entry at the NMJ (C. Thacker and T. Snutch, pers. comm.). It was predicted that the addition of upstream signaling defects to unc-2(ox8) animals should further decrease the amount of neurotransmitter released and lead to an enhancement of the locomotion defect (Figure 38). Indeed, the locomotion defect of the unc-2(0x8) single mutant was significantly enhanced (as assayed by thrashing) when both the nca-1 (gk9) and nca-2(gk9) mutations were added to the 0x8 background (Figure 33A and Table 10). Another unc-2 allele used was the putative loss-of function el29 allele that is thought to eliminate channel expression and completely remove Ca 2 + entry through UNC-2 at the NMJ (C. Thacker and T. Snutch, pers. comm.). If UNC-2 provides the only source of Ca 2 + entry necessary to trigger neurotransmitter release at the NMJ, then the addition of upstream signaling defects to unc-2(el29) animals should not affect the amount of neurotransmitter released and should not enhance the locomotion defect (Figure 38). In contrast to this notion, the locomotion defect of the unc-2(el29) mutant was significantly enhanced (as assayed by thrashing) when both the nca-1 (gk9) and nca-2(gk.9) mutations were added to the el29 background and suggests that the UNC-2 Ca 2 + channel is not the sole source of pre-synaptic Ca 2 + entry at the NMJ (Figure 33B and Table 11). This notion is supported by the finding that loss-of-function unc-2(e55) mutant animals retain a significant amount of evoked release presumably due to additional sources of Ca 2 + entry at the NMJ (Richmond etal., 2001). 244 Possible Calcium Channel Redundancy at the Cholinergic Neuromuscular Junction The possibility of other Ca 2 + channels mediating residual amounts of neurotransmitter release is supported by comparing phenotypes between unc-2 loss-of-function animals with that of cha-1 and unc-17 animals. Loss-of-function mutations in cha-1 and unc-17, which encode for choline acetyltransferase and a synaptic vesicle ACh transporter, respectively completely eliminate ACh release. Even though these mutants complete embryogenesis, they are not viable as they are paralyzed, are barely able to feed and animals die at the LI stage of development (Nonet et al., 1993; Rand and Nonet, 1997). In contrast, unc-2 loss-of-function mutant animals are viable, suggesting that even in the absence of unc-2 there still is residual release of ACh at the NMJ. Furthermore, unc-2 animals are not completely resistant to aldicarb, as they do become hypercontracted after prolonged exposure, again suggesting that UNC-2 is probably not the sole mediator of ACh release at the NMJ (Mathews et al., 2003). Given that ACh release is essential for viability in C. elegans, it is not unreasonable to speculate that there is redundancy built into the pre-synaptic system at the level of the Ca 2 + channels involved in triggering release. There are two other Ca 2 + channel genes in C. elegans that could potentially mediate neurotransmission. One of these is the putative T-type Ca 2 + channel encoded by the cca-1 gene. Cellular expression analysis of cca-1 using a promoter: :GFP reporter construct has revealed that cca-1 is expressed in a subset of motor neurons in the dorsal and ventral nerve cords but not in body wall muscle (C. Thacker and T. Snutch, pers. comm.). Previous studies have implicated T-type Ca 2 + channels in the electrical firing patterns of central neurons in vertebrates (Huguenard, 1996), as well as in neurotransmitter release from retinal bipolar cells (Pan et al., 2001). To address the possibility that the CCA-1 Ca 2 + channel provides an additional source of Ca 2 + entry at the NMJ, or is involved in signal propagation within motor neurons, various double and triple mutants between cca-1 (adl650), nca-1 (gk9) and nca-2(gk5) were generated. The cca-1 allele used was the predicted loss-of-function adl650 allele that is thought to eliminate channel 245 expression (C. Thacker and T. Snutch, pers. comm.). If the CCA-1 Ca channel provides a source of Ca 2 + entry to trigger ACh release at the NMJ, or is involved in the electrical transmission within the neuron, then a loss-of-function allele of cca-1 may lead to a locomotion defect (Figure 38). However, mutations in the cca-1 gene alone did not lead to any appreciable locomotion defect, nor did the addition of cca-1 (adl650) to the genetic background of the nca-2(gk5); nca-1(gk9) double mutant enhance its locomotion defect (Figure 34 and Table 12). Likewise, the addition of cca-1 (adl650) to the genetic background of the unc-2(ox8); nca-2(gk5); nca-1 (gk.9) triple mutant did not enhance its locomotion phenotype (Figure 35 and Table 13). Taken together, these results suggest that the CCA-1 Ca 2 + channel does not provide a significant source of Ca 2 + entry at the NMJ, nor does it seem to function similar to NCA-1 and NCA-2 in aiding in signal propagation within motor neurons. Although the pcca-l::GFP reporter construct is expressed in a subset of motor neurons in the dorsal and ventral nerve cords, the exact biological role of cca-1 within these cell types remains to be determined. Perhaps the use of a more sensitive technique than that of the thrashing assay may help to elucidate the potential roles of cca-1 in motor neurons of the dorsal and ventral nerve cords. For example, the use of automated movement analysis systems that can quantify multiple aspects of a worm's locomotory behavior on agar plates including velocity along its sinusoidal path, the degree of its dorsal-ventral flexion, the rate of wave propagation and the amplitude and wavelength of the its sinusoidal track would all help to measure subtle defects and further aid in the dissection of a gene's role in the generation of motion in C. elegans (Baek et al., 2002). The other C. elegans Ca 2 + channel that could potentially mediate neurotransmission is the putative L-type Ca 2 + channel encoded by the egl-19 gene. Although EGL-19 is thought to predominantly function as the Ca 2 + channel mediating muscle contraction (Lee et al, 1997; Jospin et al., 2002), it is also expressed in a subset of neurons (Lee et al., 1997) including motor neurons of the dorsal and ventral nerve cords (C. Thacker and T. Snutch, pers. comm.) and thus 246 may play a role in neurotransmission at the NMJ. In some vertebrate neurons, L-type Ca channels are involved in the exocytotic release of neurotransmitters and neuropeptides (Perney et al., 1986; Rane et al, 1987; Lemos and Nowycky, 1989). The hypothesis that egl-19 may contribute to ACh release at the NMJ could be tested by constructing various combinations of double, triple and quadruple mutants between nca-1, nca-2, unc-2 and egl-19 and assaying for any enhancement in the locomotion defect by the addition of egl-19 mutations to any given genetic background. Unfortunately, constructing mutant strains with egl-19 is confounded by the fact that loss-of-function alleles of egl-19 result in a Pat (paralyzed arrest at embryonic two-fold stage) phenotype (Lee et al., 1997). This Pat phenotype of loss-of-function egl-19 alleles is typical of essential muscle genes (Williams and Waterston, 1994; Moerman and Fire, 1997) and likely reflects the requirement of Ca 2 + entry through the EGL-19 Ca 2 + channel in muscle during development. In this regard, only reduction-of-function mutations in egl-19 could be used when generating the proposed mutant strains although even then it may be difficult to interpret behavioral data. For example, if it is assumed that the amount of Ca 2 + entry contributed by the EGL-19 channel is relatively small under wild-type conditions, then the corresponding change in Ca 2 + entry through a reduction-of-function mutation to the EGL-19 Ca 2 + channel may or may not be significantly reduced enough to physiologically affect movement as assayed with the thrashing assay, especially that in the unc-2(el29); nca-2(gk5); nca-1 (gk9) triple mutant strain which is already at the lower limit of the assay. Furthermore, since egl-19 has a well defined post-synaptic role in muscle contraction, it may be difficult to resolve pre-synaptic defects at the NMJ from post-synaptic defects. An additional line of experiments that could be used to explore the potential pre-synaptic role(s) of the EGL-19 Ca 2 + channel in cholinergic motor neurons could involve over-expressing egl-19 specifically in cholinergic neurons. This strategy would effectively eliminate the confounding post-synaptic effects posed by egl-19 expression in body wall muscle. In order to 247 achieve cholinergic neuron-specific expression of egl-19, the promoter of the unc-17 gene could be used as unc-17 is expressed only in cholinergic neurons (Alfonso et al., 1993). If the EGL-19 Ca 2 + channel has a pre-synaptic role in Ca 2 + entry in cholinergic motor neurons, then over-expression of a punc-17::egl-19 construct in any of the aforementioned mutant strains may partially suppress their movement defects (Figure 38). This proposed suppression would be observed if there was a significant increase in the amount of ACh released from cholinergic motor neurons at the NMJ as a result of the additional Ca 2 + influx provided by the over-expressed EGL-19 Ca 2 + channels. This proposed line of experiments assumes that any increase in cholinergic neurotransmission at the NMJ that may be supplied by the over-expression of egl-19 will result in an increase in the animals ability to move and that this change will be significantly different such that it could be detected by the thrashing assay. This experiment also makes the assumption that over-expression of EGL-19 in cholinergic neurons other than those of the dorsal and ventral nerve cords will not have an effect on locomotion. A complementary line of experiments that could be used to further explore the potential pre-synaptic role(s) of the EGL-19 Ca 2 + channel in cholinergic motor neurons would involve specifically knocking out the egl-19 gene in cholinergic neurons. This experiment would entail using the unc-17 promoter to drive egl-19 dsRNA expression (Tavernarakis et al., 2000) to knock expression out only in the cholinergic neurons and eliminate the problem posed by the Pat phenotype of loss-of-function mutations of egl-19. If the EGL-19 Ca 2 + channel has a pre-synaptic role in Ca 2 + entry in cholinergic motor neurons, then according to the hypothetical model, knockout of the egl-19 gene in cholinergic neurons in any of the aforementioned mutant strains may enhance their movement defects (Figure 38). 248 Mutations at the GABAergic Neuromuscular Junction Partially Suppresses the Locomotion Defect of the nca-2(gkS); nca-1 (gk9) Double Mutant In addition to being expressed in the cholinergic motor neurons of the dorsal and ventral nerve cords, the nca genes in C. elegans are also expressed in GABAergic neurons (Figures 26 and 27). Of particular interest is the observation that the pnca-2::GFP reporter construct was expressed in the class D GABAergic motor neurons of the dorsal and ventral nerve cords (Figures 27 and 28). Sinusoidal movement in C. elegans is generated by the out of phase contraction of dorsal and ventral body wall muscles. This feat is accomplished by the coordinated excitation of body wall muscles on one side of the animal by the excitatory class A or B cholinergic motor neurons (Alfonso et al., 1993; Driscoll and Kaplan, 1997) and the simultaneous inhibition of the body wall muscles on the opposite side by the class D inhibitory GABAergic motor neurons (Chalfie and White, 1988; Mclntire et al., 1993b) (Figure 22A). Mutations in genes involved in the function of the class D motor neurons, such as the glutamic acid decarboxylase gene unc-25, cause a "shrinker" movement phenotype due to the simultaneous hypercontraction of dorsal and ventral body wall muscles (Mclntire et al., 1993a; Jin et al., 1999). Based upon the observation that the p«ca-2::GFP reporter construct was expressed in the class D GABAergic motor neurons of the dorsal and ventral nerve cords (Figures 27 and 28) it was surprising that the nca-2(gk5) single mutant strain did not exhibit a "shrinker" phenotype or any other change to its pattern of movement that could be attributed to a defect in GABAergic neurotransmission at the NMJ. A possible explanation for the lack of any movement phenotype for the nca-2(gk5) single mutant strain is that the biological role of the NCA-2 channels in the class D motor neurons may be different from its role in cholinergic motor neurons. Another possibility is that the subcellular localization of the NCA-2 channel may be different in the two classes of motor neurons, possibly leading to cell-type specific functions of the NCA-2 channel. 249 Analysis of the synaptic connectivities of the motor neurons of the dorsal and ventral nerve cords has revealed that the class A and B cholinergic motor neurons make synapses with both the body wall muscle, as well as to the class D motor neurons that innervate the contralateral body wall muscles (White et al., 1986). Thus, mutations that affect neurotransmission at the cholinergic-GABAergic motor neuron synapse may also lead to locomotion phenotypes. Since the locomotion defect exhibited by the nca-2(gk5); nca-1 (gk9) double mutant may be due to a pre-synaptic defect in the cholinergic motor neurons and since these motor neurons make synaptic connections with the class D inhibitory motor neurons, then it is reasonable to hypothesize that there may also be defective electrical signaling within the GABAergic motor neuron circuit. Therefore, a portion of the observed locomotion defect of the nca-2 (gk5); nca-1 (gk9) double mutant may be due to altered physiology of the inhibitory motor circuit. In order to address this hypothesis, a series of different mutant strains were generated between nca-1 and/or nca-2 and unc-25(el56). The el56 allele was chosen because it is a predicted loss-of-function allele (Jin et al., 1999) and thus would be predicted to yield the most dramatic effect. As hypothesized, addition of unc-25(el56) to the genetic background of the nca-2(gk5); nca-1 (gk9) double mutant had a significant effect on the locomotion defect of the double mutant as the newly generated unc-25(el 56); nca-2(gk5); nca-1 (gk9) triple mutant moved significantly better in liquid than the nca-2(gk5); nca-1 (gk9) double mutant (Figure 36 and Table 14). This apparent suppression of the Nca-2; Nca-1 mutant phenotype by unc-25 suggests that part of the locomotion defect observed for the nca-2(gk5); nca-1 (gk.9) double mutant may be due to an altered cross-talk between the excitatory and inhibitory motor neurons that generate movement in C. elegans. According to this proposal, uncoupling of the inhibitory circuit from the excitatory circuit by loss-of-function mutations to both nca-1 and nca-2 may lead to a physiological state where 250 the ratio of excitatory to inhibitory neurotransmission is altered at the NMJ. For example, in this altered physiological state, the ventral body wall muscles may receive less excitatory cholinergic neurotransmission than in the wild-type state due to loss of NCA-1 and NCA-2 function in these neurons (Figure 38). At the same time, the contralateral dorsal body wall muscles will be receiving inhibitory GABAergic neurotransmission at unknown levels. Since the nca-2(gk5); nca-1(gk.9) double mutant does not exhibit a "shrinker" phenotype typical of mutant strains with defects in the class D GABAergic motor neurons, it is assumed that the inhibitory GABAergic pathway is not as severely impacted in the nca-2(gk5); nca-1(gk9) double mutant as is the excitatory cholinergic pathway. According to this scenario, the respective changes to the locomotory pathways would give rise to a physiological state wherein ventral body wall muscles would be chronically under excited and the contralateral dorsal body wall muscles would be inhibited and vice versa. Both of these conditions would result in decreased amounts of muscle contraction since the strength of the inhibitory pathway would exceed that of the impaired excitatory pathway and ultimately result in decreased movement. In order to confirm the proposed model for the effects of mutations at the GABAergic NMJ on the locomotion defect of the nca-2(gk5); nca-1(gk9) double mutant, additional mutant strains between nca-1, nca-2 and unc-25 reduction-of-function alleles should be generated and their ability to suppress the locomotion defect of the nca-2(gk5); nca-1(gk9) double mutant should be assessed. According to the proposed model, these unc-25 reduction-of-function alleles should suppresses the Nca-2; Nca-1 mutant phenotype to lesser degrees than the loss-of-function el 56 allele because they will not increase the effective amount of body wall muscle excitation to the same extent. In addition, electrophysiological recordings at the NMJ (Richmond et al., 1999; Richmond and Jorgensen, 1999) should be performed to measure both the amplitude of excitatory and inhibitory post-synaptic currents from both wild-type, the nca-2(gk5); nca-1(gk.9) 251 double mutant strain and all the different unc-25 alleles in the nca-2(gk5); nca-1 (gk9) double mutant background. Analysis of the Defecation Motor Program in nca Mutant Strains Failure Rate of Expulsion As mentioned previously, the nca genes are expressed in GABAergic neurons in C. elegans. While only the p«ca-2::GFP reporter construct is expressed in the GABAergic class D motor neurons of the dorsal and ventral nerve cords, both of the pnca-1 and pnca-2: :GFP reporter constructs are expressed in the GABAergic neuron DVB (Figures 26,27 and 29). Furthermore, expression of the pnca-2: :GFP reporter construct is also observed in the cells of the intestine and in the anal depressor muscle (Figure 27). Based upon the observed cellular expression patterns of these two GFP reporter constructs, it was hypothesized that mutations in nca-1 and/or nca-2 may lead to a defect in the Emc step of the defecation motor program and a corresponding increase in the failure rate of expulsion. The Emc step is the last of three sequential muscle contractions of the defecation motor program (Figure 23) and is mediated by the neurotransmitter GABA (Mclntire et al., 1993a; Mclntire et al., 1993b). In contrast to the role of GABA as an inhibitory neurotransmitter at the NMJ (Mclntire et al., 1993a; Mclntire et al., 1993b; Bamber et al., 1999; Jin et al, 1999), GABA released from the AVL and DVB motor neurons acts as an excitatory neurotransmitter to cause the simultaneous contraction of the two muscles surrounding the posterior intestine, the anal depressor and anal sphincter muscles, resulting in expulsion of intestinal contents (Beg and Jorgensen, 2003). Analysis of the defecation failure rates of the different nca mutant strains revealed the same trend as that observed for cholinergic neurotransmission at the NMJ. As was the case for locomotion, a significant change to the rate of failure was only observed in the nca-2(gk5); nca-252 I(gk9) double mutant strain (Figure 37A and Table 15). The significant increase in the expulsion failure rate observed for the nca-2(gk5); nca-1 (gk9) double mutant strain raises the possibility of functional redundancy between nca-1 and nca-2 in the DVB neuron and in the execution of the Emc step of the defecation motor program. Although the result agrees with the proposed model for the role of NCA-1 and NCA-2 in electrical signaling in neurons that make excitatory neuromuscular connections, it was surprising that the nca-2(gk5) single mutant did not exhibit an increased rate of failure as compared to wild-type as a result of its expression in the anal depressor muscle (Figure 27). Perhaps the function of the NCA-2 channel in the anal depressor muscle is not essential for ensuring their contraction under the experimental conditions used in the present study. A closer analysis of the results of the defecation assay raise a few additional points of interest. The first result of interest is the observed expulsion failure rate observed for the wild-type strain in this study. Previous analyses of the defecation motor program of C. elegans have reported that the observed failure rate for wild-type animals is routinely less than 2% (Thomas, 1990; Dal Santo et al., 1999; Mathews et al., 2003). The observed failure rate for wild-type animals in this study (9.0 +/- 2.8%) is significantly higher than that previously reported and may be the result of either an unknown mutation in the genetic background of the N2 strain that affects the Emc step of the defecation motor program, or may reflect an error in the experimental methodology. In either case, additional experiments should be performed using a different N2 strain to rule out these two possibilities before any definitive conclusions can be drawn from the data in this study. The second result of interest is that the observed rate of failure obtained for the nca-2(gk5); nca-1 (gk9) double mutant strain (27.0 +/- 6.1%) is significantly less than that previously reported for unc-2 mutants. The UNC-2 non L-type Ca 2 + channel mediating GABA release from the DVB neuron and unc-2 mutants are expulsion defective to varying degrees (Thomas, 1990; 253 Mathews et al., 2003). A study examining the expulsion failure rate of different unc-2 animals found that animals with unc-2 reduction-of-function mutations fail -44% of the time, whereas animals with unc-2 loss-of-function mutations fail -70% of the time (Mathews et al., 2003). This higher expulsion failure rate observed for either type of the unc-2 mutant is different than that seen for locomotion, where the nca-2(gk5); nca-1 (gk9) double mutant strain exhibited a more severe movement defect than the reduction-of-function unc-2(ox8) animals (see above; Mutations to both nca-1 and nca-2 Enhance the Locomotion Defects of unc-2 Mutants). This difference in the severity of the nca-2(gk5); nca-1 (gk9) double mutant phenotype as compared to the unc-2 mutants in the both the thrashing and defecation assays suggests that the NCA-1 and NCA-2 channels may play a more prominent role in electrical signaling within the cholinergic motor neurons of the dorsal and ventral nerve cords than in the GABAergic neuron DVB. Cycle Timing Interestingly, the examination of flies with mutations in the Drosophila nca homologue, Dmaiu, revealed an additional phenotype to the ones already mentioned above. Examination of the regulation of the daily locomotory rhythm of har38 and har85 mutant flies revealed that mutations to the Dmaiu gene causes an inversion of relative locomotory activity in light versus dark (Nash et al., 2002). This alteration in the diurnal behavior in the Drosophila mutants suggests that the nca family of four domain-type VGICs may be involved in the regulation of biological rhythms. The biological rhythm that has received considerable attention in C. elegans is the timing of the defecation cycle. When feeding, C. elegans has an expulsion event (Emc) every 45 to 50 seconds (Thomas, 1990; Liu and Thomas, 1994) and the timing of the cycle depends upon the oscillation of Ca 2 + levels within the cells of the intestine (Dal Santo et al., 1999). This elevation in intracellular Ca 2 + is due to release of Ca 2 + from intracellular stores through the activation of ITR-1 (IP3 receptor) and this spike in Ca 2 + subsequently leads to the 254 secretion of an unidentified messenger that helps to initiate the pBoc event (Dal Santo et al., 1999; Strange, 2003) (Figure 24). Mutations to genes expressed in the cells of the intestine including the itr-1 (IP3 receptor; Dal Santoet al., 1999), unc-43 (calmodulin kinase II; Reiner et al., 1999) and the flr-1 (degenerin/epithelial Na + channel; Take-Uchi et al., 1998) genes all disrupt the timing of the defecation cycle. Thus, the timing of the defecation cycle in C. elegans may involve the ItVmediated Ca 2 + signaling pathway, protein phosphorylation events, as well as ion channels that are involved in regulating the membrane potential of the intestinal cells (Strange, 2003). Based upon the observation that the pnca-2 :.G¥P reporter construct is expressed in intestinal cells and that the nca family of four domain-type VGICs may be activated by intracellular Ca 2 + , it was hypothesized that mutations to nca-2 may lead to a timing defect as a result of an uncoupling of Ca 2 + signaling events with that of changes to membrane potential. However, none of the nca mutant strains exhibited any significant changes to their defecation cycles (Figure 37B and Table 15) indicating that nca-2 is probably not involved in regulating the timing of this biological rhythm. The regulation of the daily locomotory rhythm in Drosophila is markedly different than the timing of the defecation cycle in C. elegans and caution must be taken when extrapolating conclusion based upon these behaviors as there may be species-specific differences in NCA channel function in regulating biological rhythms and other physiological functions. Conclusions and Future Studies The primary goal of this study was to determine the existence of four domain-type VGICs that have not been previously identified or characterized through the use of classical molecular biological and genetic techniques. The screening of the C. elegans genome and GenBank EST databases proved to be a useful means of identifying the novel nca gene family of 255 four domain-type VGICs and the sequence information gathered was used to generate full-length cDNAs for the two novel nca genes in C. elegans (nca-1 and nca-2), as well as the nca homologue from rat (rat-nca). Comparison of the predicted protein sequences of the NCA channels with that of representative Ca 2 + and Na + channel rx(i) subunits provides new information with regards to the composition of the superfamily of four domain-type VGICs. Sequence comparisons between representative Ca 2 + and Na + channels and that of the NCA channels revealed that the NCA channels likely form a distinct family of VGICs. Furthermore, a closer comparison of the well characterized S4 and P-Loop regions from Ca 2 + and Na + channel ct(i) subunits with the analogous regions from the NCA channels revealed that the nca family of four domain-type VGICs are predicted to have unique ion selectivity, activation and inactivation properties, although electrophysiological experimentation is still required to confirm these aforementioned predictions. Unfortunately, efforts to obtain functional currents that could be attributed to the NCA channels in the HEK tsA201 proved to be unsuccessful under the experimental conditions used in this study and represents an area for future research (see above; NCA Channel Expression and Electrophysiological Analysis). Electrophysiological recordings from cultured cells and brain slices have been used to provide information regarding the properties of native VGICs. The use of primary cell cultures better reflect the in vivo conditions of the particular neuron of interest and provide the native ion channels with all the necessary post-translational modifications, second messenger pathways and associations with accessory subunits required for channel function and expression. Recapitulation of these in vivo conditions necessary for channel function may not be achieved in surrogate expression systems, such as HEK tsA201 cells, and may explain why no functional NCA currents were recorded in these tissue culture cells. Pursuing native NCA currents in neuronal cell culture and/or rat brain slices may be a promising avenue of future research. The precise cellular and subcellular distribution of the NCA channel in rat brain could be determined 256 using a combination of in situ hybridization and immunohistochemical analyses (Westenbroek et al., 1992; Hell et al., 1993; Westenbroek et al., 1995; Yokoyama et al., 1995; Bourinet et al., 1999) and primary neurons can be cultured from the appropriate brain regions (Kay and Wong, 1986; Ogata and Tatebayashi, 1991). Native rat-NCA currents can be sought using whole cell patch clamp with various pulse protocols and external salines in combination with standard pharmacological agents known to block Na + and Ca 2 + channels. Complementary experiments using siPvNAs to knockdown the NCA channel expression could be utilized to confirm the identify of native rat-NCA currents. Another goal of this study was to use C. elegans as a means of exploring the biological functions of the newly identified nca gene family. The use of C. elegans in this study illustrates the strengths of a genetic approach in studying a novel gene's function and demonstrates how this approach can complement other techniques typically used in the field of ion channel biology. Loss-of-function mutations were isolated for nca-1 and nca-2 and in combination with promoter: :GFP expression data served as the basis for the rational implementation of behavioral paradigms and pharmacological assays that garnered information about the functions of the nca-1 and nca-2 genes. Through the course of experimentation, a hypothetical model was formulated which proposes that NCA-1 and NCA-2 encode non-selective cation channels that function redundantly to ensure the effective transmission of graded electrical signals within cholinergic motor neurons and that loss of both the NCA-1 and NCA-2 ion channels impairs the ability of these motor neurons to properly propagate electrical signals received from the command interneurons. This would then lead to a corresponding reduction in the amount of ACh released at the NMJ and reduced movement. According to this model, loss of both the NCA-1 and NCA-2 should also enhance additional downstream defects caused by exposure to pharmacological agents or by mutations that cause defects in neurotransmitter release. This hypothesis was 257 corroborated by the aldicarb and halothane assays, as well as by analysis of the locomotion defects exhibited by the different mutant strains generated in this study. In order to lend further support to the aforementioned hypothesis two basic sets of electrophysiological experiments could be performed. The first of these experiments would involve making electrophysiological recordings at the NMJ (Richmond et al., 1999; Richmond and Jorgensen, 1999) in both wild-type and the nca-2(gk5); nca-1 (gk9) double mutant strain in order to confirm that the locomotion defect attributed to the double mutant is in fact due to a decrease in the amount of ACh released from the cholinergic motor neurons of the dorsal and ventral nerve cords. A second set of experiments could involve making NMJ recordings from the unc-2; nca mutant strains generated in this study in order to confirm that the phenotypes observed for these different mutant strains are due to changes in the level of neurotransmitter release at the NMJ. The possibility of recording from single neurons in C. elegans by in situ patch clamp (Goodman et al., 1998) and from primary neurons in culture (Christensen et al., 2002) could both aid in the identification and electrophysiological characterization of NCA-1 and NCA-2 channels. Cells specifically expressing either nca-1 or nca-2 could be identified using the GFP and DsRed2 transgenic strains generated in this study and then introduced into wild-type or different nca mutant backgrounds. This line of experimentation would involve making recordings from the different GFP positive nca mutant strains and comparing the whole currents obtained to that of wild-type and extrapolating the component due to the NCA channel. This approach can be made even more powerful due to the observation that worms with multiple loss-of-function mutations in various VGIC genes are viable under laboratory conditions. Therefore, the "complexity" of the native whole cell currents can be reduced by the elimination of the genes and may circumvent the need for ion channel blockers when performing native recordings. This ability to eliminate gene products that may confound experimental results is not available to 258 vertebrate systems used to study VGICs and makes C. elegans an attractive system for studying native ionic currents. The hypothesis that the locomotion defect exhibited by the nca-2(gk5);nca-l(gk9) double strain is due to a signaling defect in the cholinergic neurotransmission at the NMJ may be an oversimplification of the role of nca-1 and nca-2 in contributing to locomotion in C. elegans. The movement pattern exhibited by the nca-2(gk5); nca-1 (gk.9) double mutant on seeded agar plates consists of the animal cycling between periods of inactivity and episodes of short forward or backward movement and may reflect changes to the normal pattern of locomotion achieved through cross-talk between the command interneurons at a level other than the NMJ (Chalfie and White, 1988; Zheng et al., 1999). According to the simplest model of locomotion, the two neural circuits that control movement receive inhibitory connections from one another and can alter their activity through inputs from the sensory neurons via a series of interconnected interneurons (Zheng et al., 1999; Hobert, 2003) (Figure 22B). If nca-1 and nca-2 are expressed in either the command neurons themselves or the upstream interneurons, mutations in either one of these genes may result in changes to the normal pattern of locomotion. Although the pnca-1 and pnca-2::GFP reporter constructs are not expressed in the command interneurons (data not shown), they are both expressed in a number of interneurons in the head. In this regard, they may provide excitatory or inhibitory input into the two neural circuits that control locomotion, impacting the frequency of directional change, as well as the dwell time in any given state. The first step towards exploring this alternative role for nca-1 and nca-2 in the generation and coordination of locomotion in C . elegans would be to determine the identities of the interneurons that express the pnca-1 and p«ca-2::GFP reporter constructs and to ascertain their roles in the generation of locomotion. A genetic approach can also be used to begin to address the potential alternative role of nca-1 and nca-2. Zheng et al. (1999) have found that transgenic expression of a molecularly 259 engineered dominant gain-of-function mutation in the glr-1 gene (ionotropic glutamate receptor) that is expressed exclusively in the command interneurons results in a dramatic change in locomotory behavior that is assumed to be the result of chronic excitation of the command interneurons (Zheng et al., 1999). This molecularly engineered gain-of-function mutation is a missense mutation that results in an alanine (A) to threonine (T) change in a highly conserved region in transmembrane domain III of the 52 subtype of glutamate receptor that was previously identified in the Lurcher mouse (Zuo et al., 1997; Zheng et al., 1999). When introduced into glr-1 and expressed in Xenopus oocytes, this mutation results in a chronically open GLR-1 (A/T) channel (Zheng et al., 1999). Transgenic animals (akls9) that express the altered GLR-1 (A/T) channel exhibit a locomotory behavior that is characterized by rapid switching between the forward and backward states and a corresponding increase in the frequency of reversals. While the phenotype of the akls9 transgenic strain appears somewhat similar to that of the nca-2(gk5); nca-1(gk.9) double mutant, a major difference between the two phenotypes is that the nca-2(gk5); nca-1 (gk9) double mutant strain displays long periods of inactivity between its changes in direction whereas the akls9 transgenic animal does not show this inactivity. If it is assumed that these two phenotypes can be distinguished from one another based upon the absolute amount of movement over time, then the dominant gain-of-function mutation to the glr-1 gene may prove useful in defining the primary site of the defect in the nca-2(gk5); nca-1(gk.9) double mutant strain. If the primary site of the defect in the nca-2(gk5); nca-1 (gk9) double mutant strain is at the level of the cholinergic motor neurons of the dorsal and ventral nerve cords, then this downstream defect may partially suppress the dominant gain-of-function phenotype of the akls9 transgenic strain and decrease the total amount of locomotion observed over a given period of time. Conversely, if the primary site of the defect in the nca-2(gk5); nca-1 (gk9) double mutant strain is in interneurons upstream of the command neurons, then mutations to the nca-1 and nca-2 genes may not have any deleterious effect on the downstream gain-of-function mutation in the 260 akls9 transgenic strain and fail to change the total amount of movement over a given period of time. This latter condition assumes that the defects present in the upstream interneurons in the nca-2(gk5); nca-1(gk9) double mutant strain do not have either a positive or negative affect on the level of activity in the command interneurons and thus would not enhance or suppress the phenotype of the akls9 transgenic strain. Interestingly, a "similar" type of locomotion defect to that exhibited by the nca-2(gk5) ; nca-1(gk9) double mutant in C. elegans is also exhibited by flies that have mutations in the Drosophila nca homologue, Dmaiu- Mutations in Dmaiu results in a "hesitant walking" phenotype which is characterized by a tendency for the flies to walk in an atypical manner which consists of walking for a few steps, stopping and then walking again (Krishnan and Nash, 1990). Examination of the protein expression pattern of the Drosophila NCA channel (Dmaiu) in fly heads using an antibody directed against the channel revealed high channel expression in the lateral triangles of the ellipsoid body in the fly brain (Nash et al., 2002). Previous work on walking behavior in Drosophila has determine that the ellipsoid body is one of the structures responsible for the generation and coordination of locomotion in flies (Strauss and Heisenberg, 1993; Renn et al., 1999). It is conceivable that mutations to an ion channel protein expressed in such a brain region may lead to an altered pattern of movement. 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