"Medicine, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Mathews, Eleanor Alexandra"@en . "2009-07-27T22:36:29Z"@en . "2000"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "Voltage-gated calcium channels (VGCCs) play a central role in a number of biological\r\nprocesses, including neurotransmitter release, excitation-contraction coupling, regulation of\r\ngene expression, and neuronal migration. In addition, these proteins have been implicated\r\nin a number of diseases such as epilepsy, migraine headaches, and cardiovascular illness.\r\nWhile VGCCs have been the subject of a great deal of research aimed at further clarifying\r\ntheir role in these processes, a genetic approach would strongly complement the molecular\r\nand electrophysiological approaches currently utilized to study VGCCs. In an effort to\r\ndevelop such a system, I cloned a VGCC a, subunit from the nematode Caenorhabditis\r\nelegans. This a, subunit, later shown to correspond to the unc-2 locus (Schafer and\r\nKenyon, 1995), is most closely related to the N- and P/Q-type \u00CE\u00B1\u00E2\u0082\u0081 subunits expressed in the\r\nmammalian nervous system.\r\nTo isolate new alleles of unc-2, which would allow me to identify residues\r\nimportant for VGCC function, I carried out a precomplementation screen. Eleven new\r\nalleles were identified in this screen, and several additional alleles were generously\r\nprovided by E. M. Jorgensen and J. B. Rand. I identified the corresponding sequence\r\nalterations in eight of these alleles: mdl064, mdll86, md328, ra605, ra610, ra611, ra612\r\nand ra614. Two alleles, mdl!86 and mdl064, were isolated in a mutator background\r\n(Miller et al., 1996) and have Tel transposon insertions in domains IS3 and IVS4,\r\nrespectively. ra605 and ra610 are nonsense mutations that probably eliminate unc-2\r\nfunction. Similarly, md328 is a complex rearrangement that also is expected to be null.\r\nThe ra614 allele was isolated in a reversion screen of unc-2(mdl064), and contains a\r\ntyrosine to cysteine alteration at the site of the mdl064 Tel insertion. Finally, ra611 and\r\nra612 are missense mutations in the voltage-sensing region of domain IV and the carboxyl\r\nterminus, respectively.\r\n\r\nThe behavioral defects in these mutants were analyzed using locomotion and\r\ndefecation assays. Locomotion is primarily mediated by acetylcholine (ACh), while the\r\nexpulsion step of the defecation cycle is mediated by GAB A. The mutants exhibited strong\r\ndefects in both behaviors, although ra612 homozygotes were significantly less impaired\r\nthan the null mutants. In addition, the unc-2 mutants were found to be resistant to the\r\nacetylcholinesterase (AChE) inhibitor aldicarb while exhibiting a normal response to the\r\nACh agonist nicotine. Together, these results implicate unc-2 in both cholinergic and\r\nGABAergic neurotransmission.\r\nBoth ra611 and ra.612 alter glycine residues that are conserved amongst all at\r\nsubunits cloned to date, suggesting that these mutations might alter the functional properties\r\nof the a, subunit. The corresponding alterations were introduced into the rat brain \u00CE\u00B1\u00E2\u0082\u0081[sub A]\r\n(P/Q-type) and \u00CE\u00B1\u00E2\u0082\u0081[sub B] (N-type) subunits, and expressed in HEK cells to assay their effects on\r\nthe electrophysiological properties. The ra612 mutation was found to shift the voltage\r\ndependence of activation to more depolarized potentials while also shifting the voltagedependence\r\nof inactivation in a hyperpolarized direction. Furthermore, the rate and\r\nmagnitude of current inactivation were dramatically increased by the mutation. In contrast,\r\nthe ra611 mutation did not appear to significantly alter the electrophysiological properties of\r\nthe channel, although it was associated with decreased whole cell currents. Taken together,\r\nfindings suggest that C. elegans is a powerful tool for studying VGCC structure, function,\r\nand physiology.\r\niii"@en . "https://circle.library.ubc.ca/rest/handle/2429/11351?expand=metadata"@en . "24471807 bytes"@en . "application/pdf"@en . "MOLECULAR AND GENETIC ANALYSIS OF THE UNC-2 VOLTAGE-GATED CALCIUM CHANNEL IN CAENORHABDITIS ELEGANS. by ELEANOR ALEXANDRA MATHEWS B.Sc, McGill University, 1991 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Neuroscience Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 2000 \u00C2\u00A9 Eleanor Alexandra Mathews, 2000 UBC Special Collections - Thesis Authorisation Form http://www.library.ubc.ca/spcoll/thesauth.html I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h C olumbia Vancouver, Canada 1 of 1 07/17/2000 3:44 PM Abstract Voltage-gated calcium channels (VGCCs) play a central role in a number of biological processes, including neurotransmitter release, excitation-contraction coupling, regulation of gene expression, and neuronal migration. In addition, these proteins have been implicated in a number of diseases such as epilepsy, migraine headaches, and cardiovascular illness. While VGCCs have been the subject of a great deal of research aimed at further clarifying their role in these processes, a genetic approach would strongly complement the molecular and electrophysiological approaches currently utilized to study VGCCs. In an effort to develop such a system, I cloned a VGCC a, subunit from the nematode Caenorhabditis elegans. This a, subunit, later shown to correspond to the unc-2 locus (Schafer and Kenyon, 1995), is most closely related to the N- and P/Q-type a, subunits expressed in the mammalian nervous system. To isolate new alleles of unc-2, which would allow me to identify residues important for VGCC function, I carried out a precomplementation screen. Eleven new alleles were identified in this screen, and several additional alleles were generously provided by E. M. Jorgensen and J. B. Rand. I identified the corresponding sequence alterations in eight of these alleles: mdl064, mdll86, md328, ra605, ra610, ra611, ra612 and ra614. Two alleles, mdl!86 and mdl064, were isolated in a mutator background (Miller et al., 1996) and have Tel transposon insertions in domains IS3 and IVS4, respectively. ra605 and ra610 are nonsense mutations that probably eliminate unc-2 function. Similarly, md328 is a complex rearrangement that also is expected to be null. The ra614 allele was isolated in a reversion screen of unc-2(mdl064), and contains a tyrosine to cysteine alteration at the site of the mdl064 Tel insertion. Finally, ra611 and ra612 are missense mutations in the voltage-sensing region of domain IV and the carboxyl terminus, respectively. ii The behavioral defects in these mutants were analyzed using locomotion and defecation assays. Locomotion is primarily mediated by acetylcholine (ACh), while the expulsion step of the defecation cycle is mediated by GAB A. The mutants exhibited strong defects in both behaviors, although ra612 homozygotes were significantly less impaired than the null mutants. In addition, the unc-2 mutants were found to be resistant to the acetylcholinesterase (AChE) inhibitor aldicarb while exhibiting a normal response to the ACh agonist nicotine. Together, these results implicate unc-2 in both cholinergic and GABAergic neurotransmission. Both ra611 and ra.612 alter glycine residues that are conserved amongst all a t subunits cloned to date, suggesting that these mutations might alter the functional properties of the a, subunit. The corresponding alterations were introduced into the rat brain a 1 A (P/Q-type) and a 1 B (N-type) subunits, and expressed in HEK cells to assay their effects on the electrophysiological properties. The ra612 mutation was found to shift the voltage dependence of activation to more depolarized potentials while also shifting the voltage-dependence of inactivation in a hyperpolarized direction. Furthermore, the rate and magnitude of current inactivation were dramatically increased by the mutation. In contrast, the ra611 mutation did not appear to significantly alter the electrophysiological properties of the channel, although it was associated with decreased whole cell currents. Taken together, findings suggest that C. elegans is a powerful tool for studying VGCC structure, function, and physiology. iii Table of Contents ABSTRACT ii TABLE OF CONTENTS \v LIST OF FIGURES \* LIST OF TABLES xi LIST OF ABBREVIATIONS x\i ACKNOWLEDGMENTS xV DEDICATION x v i CHAPTER 1. INTRODUCTION 1 CALCIUM-MEDIATED SIGNALING IN NORMAL CELLULAR PROCESSES AND PATHOLOGICAL CONDITIONS. 1 CLASSIFICATION OF VGCCs 2 Low- Voltage Activated (T-type) Channels 3 High-Voltage Activated Ca2+ Channels 6 L-type Channels 6 N-type channels 10 Other HVA VGCCs: P-, Q-, and O-type channels 17 R-type current. 22 HVA Ca2+ Channels are Multiprotein Complexes 22 Structure and properties ofVGCC a, subunits 23 Class A 30 Class B 32 iv Class C 35 Class D 39 Class E 42 Class F 45 Classes G, H, and 1 46 Structure and properties of the auxiliary VGCC subunits 49 The p Subunit 49 The C\u00C2\u00A328 subunit 51 The y subunit 52 Modulation 53 Phosphorylation 55 G-Proteins 57 Structure and Function Studies 59 Project Rationale: The need for a genetic system to study VGCCs 62 CHAPTER 2. METHODS AND MATERIALS 70 GENETICS. 70 Nematode strains and growth conditions 70 Identification of new unc-2 alleles 71 Construction of egl-19; unc-2 and unc-36; unc-2 double mutants 71 Localization of Tel insertion sites in mdl064 and mdll86. 72 Isolation of Tel revertants 74 Phenotypic analysis of unc-2 mutants 74 Aldicarb and Nicotine Resistance 74 Thrashing Assay 74 Expulsion Assay 75 MOLECULAR BIOLOGY. 75 v PCR amplification procedures 75 R T - P C R 75 P C R 76 5' RACE 77 Genomic DNA Preparation and Amplification 78 RNase Protection Assay 78 Cycle Sequencing of PCR Products 82 Screening of the XACT-RB2 cDNA library 83 CONSTRUCTS GENERATED IN THIS STUDY. 85 cDNA Clones 85 Genomic Clones: 86 GENERATION OF MUTANT CONSTRUCTS 86 aIB Clones 86 a 1 B -612 87 a 1 B-611 88 a1A Clones 89 ma 1 A -612 89 GENERATION OF ANTIBODIES AND IMMUNOHISTOCHEMISTRY 90 Fusion Protein Construct: 90 Fusion protein expression, purification, and analysis 91 Generation of polyclonal antisera 92 Western blotting 92 Immunofluorescence staining 93 ELECTROPHYSIOLOGICAL RECORDINGS FROM H E K TSA201 CELLS. 93 Protocols for analysis of a1A constructs 94 Protocols for analysis of a1B constructs 97 Data analysis 97 vi C H A P T E R 3. T H E UNC-2 G E N E E N C O D E S A N O N - L - T Y P E V G C C a, SUBUNIT. 99 Background. 99 Results. 101 Cloning and sequencing of a VGCC a1 subunit in C. elegans 101 The complete genomic structure of the unc-2 gene 112 The primary structure of the UNC-2 VGCC oc, subunit. 115 UNC-2 expressed throughout development in C. elegans 135 C H A P T E R 4. GENETIC A N D PHENOTYPIC ANALYSIS OF UNC-2 A L L E L E S . . . . 138 BACKGROUND. 138 RESULTS. 139 Isolation of new mutations in the unc-2 gene 139 Three mutations result in putative null alleles of unc-2 142 Two unc-2 mutations are single amino acid substitutions 144 Two alleles of unc-2 are transposon-induced 144 Interactions between unc-2 and other genes involved in Ca2+-mediated signaling 154 Phenotypic analysis of unc-2 alleles 156 C H A P T E R 5. ELECTROPHYSIOLOGICAL ANALYSIS OF A L T E R E D a, SUBUNITS IN A H E T E R O L O G O U S EXPRESSION S Y S T E M 166 BACKGROUND. 166 RESULTS 167 The unc-2(ra612) mutation (G1442R) alters the temporal course of the P/Q-type current. 167 G1442R produces a positive shift in the current-voltage relationship 174 G1442R shifts the steady-state inactivation to more negative potentials 174 v i i The effects of the G1442R mutation on channel properties are independent of the charge carrier. 179 The unc-2(ra611) mutation (G1254R) does not appear to affect the electrophysiological properties of exogenously expressed a1B channels 179 CHAPTER 6. DISCUSSION. 183 Molecular cloning of a VGCC a1 subunit in the nematode C. elegans 183 Genomic organization of the VGCC a, subunit encoded by the unc-2 gene 184 unc-2 likely encodes an HVA, DHF'-insensitive VGCC. 185 The UNC-2 VGCC a, subunit appears to be expressed as a single isoform 187 Isolation and characterization of novel mutations in the unc-2 locus 188 UNC-2 is involved in acetylcholine and GAB A neurotransmission 190 UNC-2 and UNC-36 may function in the same VGCC complex 192 Analysis of unc-2 mutations in a heterologous expression system 195 The ra612 mutation alters a number ofP/Q-type channel properties 195 The fast inactivation seems to be a property of the OC1A_6]2 subunit. 197 The increased rate of inactivation does not appear to be Ca2+-dependent. 198 Future studies on the unc-2(ra612) mutation 200 The ra611 mutation does not alter the basic electrophysiological properties of the a1B N-type channel 202 GENERAL DISCUSSION - C A 2 + CHANNELS IN SYNAPTIC TRANSMISSION 205 A general mechanism of neurotransmitter release 206 The mechanism of neurotransmission is highly conserved. 208 UNC-2 may provide the Ca2+ signal in C. elegans nervous transmission 210 Conclusion and future studies 212 R E F E R E N C E S 214 viii List of Figures FIGURE 1. COMPOSITION OF A V G C C COMPLEX AND STRUCTURE OF THE a, SUBUNIT 25 FIGURE 2. SIMILARITY TREE OF MAMMALIAN V G C C a, SUBUNITS 29 FIGURE 3. DIAGRAM OF THE RNASE PROTECTION ASSAY 80 FIGURE 4. PROTOCOLS FOR ELECTROPHYSIOLOGICAL ANALYSIS 96 FIGURE 5. ALIGNMENT OF THE CE2 SEQUENCE WITH THE HOMOLOGOUS REGION OF OTHER VOLTAGE-GATED NA + AND C A 2 + CHANNELS 103 FIGURE 6. GENOMIC SOUTHERN BLOT OF CE2/UNC-2 106 FIGURE 7. GENOMIC LOCALIZATION OF CE2/UNC-2 108 FIGURE 8. SOUTHERN BLOT OF COSMID T02C5 110 FIGURE 9. STRUCTURE OF THE UNC-2 GENE AND PROTEIN PRODUCTS 114 FIGURE 10. COMPLETE AMINO ACID SEQUENCE OF THE U N C - 2 V G C C a, SUBUNIT 117 FIGURE 11. SIMILARITY TREE OF V G C C a; SUBUNITS 122 FIGURE 12. ALIGNMENTS OF U N C - 2 AMINO ACID SEQUENCE WITH MAMMALIAN DFTP-INSENSITIVE V G C C S 124 FIGURE 13. ALIGNMENT OF REGIONS OF UNC-2 WITH THE ANALOGOUS REGIONS OF THE MAMMALIAN H V A V G C C 132 FIGURE 14. NORTHERN BLOT OF STAGED C. ELEGANS TOTAL R N A 137 FIGURE 15. FLOWCHART ILLUSTRATING THE PRECOMPLEMENTATION SCREEN USED TO ISOLATE NOVEL ALLELES OF UNC-2 141 FIGURE 16. TYPICAL EXAMPLE OF POSITIVE RESULTS FROM R P A SCREEN 146 FIGURE 17. LOCATION OF MUTATIONS IN UNC-2 148 FIGURE 18. DIAGRAM ILLUSTRATING THE P C R STRATEGY FOR LOCALIZING THE TC 1 ELEMENTS IN MD1064 AND MD1186 153 FIGURE 19. THRASHING ANALYSIS OF THE UNC-2 ALLELES 161 FIGURE 20. DEFECATION ANALYSIS OF THE UNC-2 ALLELES 164 ix FIGURE 21. THE G1442R MUTATION AFFECTS THE TEMPORAL COURSE OF THE P/Q-TYPE CURRENT AND MODIFIES THE VOLTAGE-DEPENDENCE OF NON-INACTTVATING CURRENT 169 FIGURE 22. THE G1442R MUTATION ALTERS THE CURRENT-VOLTAGE RELATIONSHIP AND STEADY-STATE INACTTVATION PROPERTIES OF THE CHANNEL 173 FIGURE 23. a 1 A . 6 1 2 C A 2 + CURRENTS DISPLAY SIMILAR PROPERTIES 176 FIGURE 24. G1442R MODIFIES THE VOLTAGE DEPENDENCE OF THE FRACTION OF NON-INACTIVATING CURRENT (/?) 178 FIGURE 25. THE Gl 254R MUTATION DOES NOT ALTER THE MACROSCOPIC A 1 B . N CURRENT 181 x List of Tables TABLE 1. V G C C TYPES 4 TABLE 2. MUTATIONS IN V G C C SUBUNIT GENES 63 TABLE 3. GENETICALLY-DEFINED VOLTAGE-GATED ION CHANNEL GENES IN C. ELEGANS 67 TABLE 4. SEQUENCE OF OLIGONUCLEOTIDE PRIMERS 73 TABLE 5. AMINO ACID % IDENTITY/SIMILARITY BETWEEN V G C C a, SUBUNITS 120 TABLE 6: MUTANT ALLELES OF UNC-2 143 TABLE 7. MOLECULAR ALTERATIONS IN UNC-2 143 TABLE 8. SUMMARY OF ALDICARB AND NICOTINE RESISTANCE 159 TABLE 9. SUMMARY OF THE THRASHING AND DEFECATION RATES OF MUTANT STRAINS 159 TABLE 10. SUMMARY OF THE PROPERTIES OF a, A AND a 1 A . 6 ] 2 171 x i List of Abbreviations 5-HT serotonin (5-hydroxytryptamine) ACh acetylcholine AChE acetylcholinesterase AID a, subunit interaction domain Ba 2 + barium BAFTA 1,2-bjs(2-aminop_henoxy)ethane N,N,N', N'-tetraacetate BID 15 subunit interaction domain bp basepairs Ca 2 + calcium Cd 2 + cadmium cG-PK cGMP-dependent protein kinase CHO Chinese hamster ovary CNS central nervous system DHP dihydropyridine dNTPs deoxyribonucleoside triphosphates dpy dumpy DRG dorsal root ganglion DTT dithiothreitol E-C excitation-contraction EDTA disodium ethylenediaminetetraacetate EGTA ethylene glycol-bis(aminoethylether)N,N,N', N'-tetraacetate EtBr Ethidium Bromide FTX funnel web spider toxin G-protein heterotrimeric guanyl-nucleotide-binding-proteins GABA y-aminobutyric acid xii Gd 3 + gadolinium GST glutathione S-transferase HEK human embryonic kidney HEPES N-2-hydroxyethylpjperazine-N'-2-ethanesulfonic acid HVA high voltage-activated i c 5 0 concentration of compound that produces 50% inhibition I-V relationship current-voltage relationship IPTG isogropylthio-P-Dgalactoside kb kilobase LVA low-voltage-activated mdg muscular dysgenesis ml milliliter mM millimolar ms millisecond mV millivolt mil mega ohms MT-PBS mouse tonicity-phosphate buffered saline NA noradrenaline Na+ sodium ng nanogram Ni 2 + nickel NMJ neuromuscular junction co-Aga oo-agatoxin co-CTx CO-conotoxin G>CgTx oo-conotoxin GVIA pA picoAmps PAGE polyacrylamide gel electrophoresis xiii PBS phosphate-buffered saline PCR polymerase chain reaction PKA cAMP-dependent protein kinase PKC protein kinase C pm picomole PMSF phenylmethylsulfonylfluoride PNS peripheral nervous system pS pico seimens PTX pertussis toxin ric resistant to inhibitors of cholinesterases rpm revolutions per minute RT-PCR reverse transcription-polymerase chain reaction SDS sodium dodecyl sulfate sFTX synthetic funnel-web spider toxin Sr2 + strontium SS i n a c t steady-state inactivation TBS Tris-buffered saline TBS-T Tris-buffered saline plus Tween-20 TEA tetraethylarnmonium i time constant LLI microliter (ig microgram unc uncoordinated VGCC voltage-gated calcium channel xiv Acknowledgments I would like to express my sincerest thanks to my supervisor Dr. Terrence Snutch, for his patience, support, and guidance. I would also like to thank members of the Snutch lab, past and present, including Dr. Esperanza Garcia, Dr. Celia Santi, Dr. Donald Nelson, and Dr. Anthony Stea. In addition, I would like to express my gratitude to Dr. Donald Moerman and members of his lab for introducing me to C. elegans biology and for innumerable contributions to the success of this project, and to Dr. David Baillie for his assistance in identifying the 5' end of the unc-2 gene. Finally, I would like to thank my friends, colleagues, and mentors at the University of British Columbia, including Dr. Jacob Hodgeson, and my supervisory committee: Dr. Linda Matsuuchi, Dr. Steve Vincent, and Dr. Cathy Rankin. xv Dedication This thesis is dedicated to my husband, Gregory P. Mullen, without whose help and support it would not have been possible. xvi Chapter 1. Introduction, Calcium-mediated signaling in normal cellular processes and pathological conditions. Calcium (Ca2+) entry into cells mediates a number of physiological functions including activation of Ca2+-dependent enzymes, modulation of ion channel activity, neurotransmitter release, regulation of gene expression, cell migration, and muscle contraction (Tanabe et al, 1988; Catterall, 1991; Komuro and Rakic, 1992; Uchitel et al, 1992; Bading et al, 1993; Wheeler et al, 1994; reviewed in Levitan, 1999). Rapid Ca 2 + influx is mediated by integral membrane proteins called voltage-gated calcium channels (VGCCs). VGCCs respond to membrane depolarization with a change in conformation which opens a Ca 2 +-selective pore, allowing Ca 2 + to enter the cell. The amount and timing of Ca 2 + influx can be directly modulated by protein kinases and phosphatases, as well as by neurotransmitters and hormones acting on the channels through a variety of second messenger pathways (reviewed in Catterall, 1991; Snutch and Reiner, 1992). In addition to the requirement for Ca 2 + in normal cellular functioning, excess intracellular Ca 2 + appears to be involved in a number of pathological states (Janis and Triggle, 1991; Peters et al, 1991). Excessive Ca 2 + influx is implicated in neurotoxicity and cell death. In addition, diseases such as angina, hypertension, stroke, and certain 1 arrythmias are treated with compounds that directly affect either VGCC or proteins that modulate their activity. Research on VGCCs will lead to a better understanding of channel structure and function, as well as identifying interacting and modulatory proteins. Understanding the structural and functional components of VGCC signaling will also provide insight into the roles that these molecules play in cardiovascular and neurological disorders and facilitate the development of therapeutic compounds specific for individual VGCC subtypes. A living system in which physiologically relevant mutations can be easily generated, isolated, and identified will greatly facilitate VGCC structure-function analysis. In this thesis, I used a model system to study VGCCs by cloning and characterizing a VGCC a, subunit in the nematode Caenorhabditis elegans. I generated mutations in the gene and examined their effects at functional and behavioral levels. This approach complemented those currently used to study mammalian VGCCs and provided new information concerning the structure, function, and regulation of the channel. Classification of VGCCs. Electrophysiological recordings from neurons, muscle, and endocrine cells revealed Ca 2 + currents with distinct characteristics, indicating the existence of multiple types of VGCCs. Early studies on muscle cells identified two classes of VGCCs, low-voltage activated (LVA) and high-voltage activated (HVA), based on the membrane potentials at which they first open (reviewed in Jones, 1998). The LVA channels, which typically have a small conductance (8-10 pico Siemens (pS)), open in response to small changes from the resting membrane potential and inactivate rapidly. The LVA channels are also referred to as \"T\" (tiny), \"low-threshold\", or \"fast\" VGCCs. In contrast, HVA currents are activated by stronger depolarizations and display variable inactivation kinetics. In muscle, the channels carrying the HVA current have a unitary conductance of 20-25 pS, hence the names \"L\" 2 (large or long-lasting), \"high-threshold\", or \"slow\" VGCCs. Single channel recordings in neurons indicated that in addition to the L-type channel, a second channel, designated N-type, contributes to the HVA-activated current (Nowycky et al, 1985). Subsequent studies identified several additional HVA VGCCs with properties distinct from those of either N- or L-type channels. Members of this family of VGCCs are categorized on the basis of a number of criteria including single channel conductance, kinetics, pharmacology, and cellular distribution (Nowycky et al, 1985; reviewed in Bean, 1989; Scott et al, 1991; andTsien etai, 1991) (Table 1). Low-Voltage Activated (T-type) Channels. LVA (T-type) channels were first described in rat and chick sensory neurons (Carbone and Lux, 1984; Nowycky et al, 1985), but also are present in other excitable tissues, including cardiac sinoatrial cells, smooth and developing skeletal muscle, neuroendocrine cells, and thalamic neurons, as well as non-excitable cells such as fibroblasts, osteoblasts, and astrocytes (Miller, 1987; Bean, 1989; Tsien, et al, 1991). Other cell types, such as sympathetic neurons, superior cervical ganglion cells, and adrenal chromaffin cells, appear not to express significant T-type currents (Bean, 1989; Bean and McDonough, 1998). As described above, T-type channels are activated by relatively small depolarizations from hyperpolarized membrane potentials. Typically, these channels first open at potentials positive to -70 mV and whole-cell currents are maximal at approximately -40 mV. T-type channels are usually fully inactivated at resting potentials greater than -40 mV. While T-type channels inactivate rapidly in a voltage-dependent manner, tail current measurements indicate that T-type channels deactivate, or close, slowly. Because these channels are inactivated at positive holding potentials, very negative holding potentials (-80 mV or more negative) are required for full availability of the channels. The kinetics of activation and inactivation also display voltage dependency; the rates are slow near 3 Table 1. VGCC Types. Channel Type Subunit Activation Range Pharmacological Sensitivities Localization P/Q Class A HVA Cd 2 + , co-Aga IVA (P), co-MVHC (Q); insensitive to DHPs CNS (especially in cerebellum), heart, pituitary; dendrites and presynaptic terminals N Class B HVA co-CgTx (irreversible), Cd 2 + , insensitive to DHPs nervous system; dendrites and presynaptic terminals L Classes C, D, F*, and S HVA Cd 2 + , CO-CgTx (reversible; a 1 D ) , DHPs, benzothiazapines, phenylalkylamines oclc: smooth and cardiac muscle; CNS a , D : CNS, endocrine a 1 F: retina a, s: skeletal muscle; (oclc, a 1 D) cell bodies and proximal dendrites R Class E rvA Cd 2 + , Ni 2 +; insensitive to DHPs, co-CgTx, and u)-Aga IVA CNS; cell bodies and some distal dendrites T Classes G, H, and I LVA mibefradil, amiloride CNS, heart, placenta, lung, and kidney *= inferred from protein sequence 4 threshold potentials and accelerate with increasing potentials (Nowycky et al., 1985; Fox et al., 1987a). The study of T-type channels has lagged behind that of other VGCCs, in part due to the lack of cDNA clones representative of this type (see below), but primarily because of the lack of pharmacological agents that selectively affect T-type VGCCs. T-type channels are sensitive to the divalent cations nickel (Ni2+), cadmium (Cd2+), and zinc (Zn2+), with Ni 2 + being the most potent. However, in some cell types, low concentrations of these cations fail to block T-type currents or block other non-LVA Ca 2 + currents (reviewed in Huguenard, 1996; Zamponi et al., 1996). A number of organic compounds inhibit T-type channels, but often at concentrations that block other VGCCs. Octanol and the sodium (Na+) channel blocker amiloride have been utilized as T-type channel antagonists, although these compounds also inhibit some components of the HVA current (Bean, 1989; reviewed in Tsien et al., 1991; and Huguenard, 1996; Bean and McDonough, 1998; McDonough and Bean, 1998). The antihypertensive, mibefridil, may be the most selective T-type channel blocker identified to date. Nanomolar concentrations of mifebradil almost completely block T-type current while reducing Ca 2 + currents through other channel types by less that 5% (McDonough and Bean, 1998). Perhaps the most interesting class of compounds that inhibit T-type VGCCs are the anticonvulsant drugs. Ethosuximide, a drug used to treat absence epilepsy, has been shown to reduce current through T-type channels with little effect on HVA channels (Huguenard, 1996; Bean and McDonough, 1998). While direct evidence linking T-type channels to specific physiological roles is limited, their electrophysiological profiles and cellular and subcellular localizations suggest a number of possible functions. T-type channels are found in many cells that display spontaneous electrical activity, such as sinoatrial nodal cells of the heart, neuroendocrine cells, and thalamic neurons. This expression profile, together with their low threshold of activation, suggests that T-type channels play a role in pacemaker activity. Localization of T-type channels in dendritic branches has also led to the suggestion that initiation of low-5 threshold Ca + spikes may serve to amplify incoming signals. The low activation threshold, along with the requirement for hyperpolarized membrane potentials to overcome inactivation of these channels, has also led to speculation that T-type channels are involved in generating the oscillatory firing in some neurons. T-type channels may also exert an effect by generating a steady inward current which could mediate the gating of Ca 2 +-dependent ion channels and regulate Ca2+-dependent enzymes and gene expression. Finally, T-type currents are present in developing muscle and nervous tissue, suggesting that these channels may play a developmental role (Beam and Knudson, 1988[a,b]; Bean, 1989; Kostyuk, 1989). High-Voltage Activated Ca 2 + Channels. The HVA Ca 2 + current was originally defined as a non-inactivating Ca 2 + current that activated upon large membrane depolarizations. However, electrophysiological studies on neuronal cells indicate that the HVA current can be further resolved to include N-, P-, and Q-type channels along with the L-type. These channels all require significant depolarizations, generally in the range of -30 mV, to become activated. However, they differ with respect to kinetics, single channel conductance, and pharmacology, and it is on the basis of these criteria that they are distinguished (Nowycky et al, 1985; reviewed in Bean, 1989; Tsien etai, 1988, 1991; Scott etai, 1991). L-type Channels. L-type VGCCs were initially described in peripheral neurons and cardiac cells, but appear to be present in all excitable and many non-excitable cells (Tsien et al, 1991). The L-type channel is the primary route for Ca 2 + entry into cardiac, skeletal, and smooth muscles (Bean, 1989). In certain cells, L-type channels are localized to specific subcellular regions. 6 For example, the L-type channels responsible for skeletal muscle contraction are concentrated on the transverse tubule membrane (Tanabe et al, 1987), while neuronal L-type channels are located primarily on cell bodies and proximal dendrites (Hell et al, 1993a). L-type channels have a unitary conductance ranging from 20 and 27 pS using 110 mM barium (Ba2+) as the charge carrier. L-type channels require large departures from resting potential to become activated and typically begin to open at potentials positive to -10 mV, although they can activate at significantly more negative potentials in chromaffin cells, sensory neurons, and cardiac cells. Once open, these channels do not inactivate significantly during depolarizations of hundreds of milliseconds (reviewed in Tsien et al, 1988; Bean, 1989; Scott et al, 1991). Compared to Ba 2 + currents, the electrophysiological profile of L-type channels is different when Ca 2 + is used as the charge carrier. Under these conditions, L-type currents are smaller and inactivate rapidly over time. Ca2+-dependent inactivation has a number of characteristic properties (reviewed in Imready and Yue, 1994). Ca 2 + currents decay rapidly, while currents carried by Ba2 +, strontium (Sr2+), and Na+ ions are much longer-lasting. In addition, inactivation attributable to Ca 2 + influx is greatest at depolarizations at which Ca 2 + entry through the channel is maximal. While the degree of inactivation is slowed by the addition of B APT A and other Ca 2 + chelators, it is not completely abolished. However, Ca2+-dependent inactivation can be eliminated by intracellular applications of trypsin, suggesting that the mechanism through which Ca 2 + acts to inactivate the channel is in close proximity to, if not part of, the channel complex itself (reviewed in Bean, 1989; and Scott et al, 1991; Charnet et al, 1994; Neely et al, 1994). Because the rate of Ca 2 +-dependent inactivation does not change with channel density, Neely et al. (1994) proposed a \"local domain\" hypothesis, in which Ca 2 + affects only the channel through which it enters. 7 A number of models to describe the mechanism of Ca2+-dependent inactivation have been proposed. One postulates that this inactivation is the result of a Ca2+-activated second messenger system involving dephosphorylation of the a, subunit (Chad and Eckert, 1986). For example, calcineurin, a Ca2+-calmodulin-dependent phosphatase, had been proposed as a potential candidate based on the finding that intracellular application increased the rate of Ca2+-dependent inactivation. The direct action of calmodulin on the channel had also been suggested (reviewed in Scott et al, 1991; reviewed in Imready and Yue, 1994). Imready and Yue (1994) initially discounted both of these hypotheses by showing that compounds that abolish all known phosphatase activity do not eradicate Ca2+-dependent inactivation. Likewise, calmodulin inhibitors had no affect on Ca2+-dependent inactivation. Instead, these authors propose a model in which Ca 2 + binds directly to the channel, inducing a conformational change that favors channel closing. They suggest two regions in which this may occur; the carboxyl tail of the channel, which contains a putative EF hand (a Ca 2 +-binding motif), and the 11-111 linker, which is rich in negatively-charged residues. Subsequent studies have implicated the EF-hand region as well as other portions of the carboxyl tail in Ca2+-dependent inactivation (de Leon et al, 1995; Zhou et al, 1997; Soldatov etal, 1997; Soldatov etal, 1998; Ziihlke and Reuter, 1998). However, several recent studies have demonstrated that calmodulin probably acts as the sensing mechanism that triggers Ca2+-dependent inactivation (Peterson et al, 1999; Ziihlke et al, 1999). Much is known about the pharmacological properties of L-type VGCCs largely because of the development of drugs used in the treatment of cardiovascular diseases (reviewed in Miller, 1987; Bean, 1989; and Scott et al, 1991). The three main classes of organic L-type channel blockers are phenylalkylamines (verapamil), benzothiazapines (diltiazem), and 1,4-dihydropyridines (DHPs) (nitrendipine, nifedipine, nimodipine). The DHPs have the highest affinity for L-type channels and interact in a voltage-dependent manner (producing more potent inhibition at depolarized potentials). Voltage-dependency arises because DHPs bind preferentially to channels in the active conformation, a state 8 favored by depolarization. In addition to the DHP antagonists, a number of DHP agonists have been developed, the most common of which is (-)-Bay K 8644. These compounds increase both the open time and the single channel conductance. L-type VGCCs also appear to be highly sensitive to block by Cd 2 + and Ni 2 +. The peptide toxin co-agatoxin UIA (co-Aga IIIA), isolated from the venom of the funnel web spider Agelenopsis aperta, is a potent inhibitor of L-type VGCCs in myocytes and neurons (Mintz et al, 1991; Cohen et al, 1992). co-Aga IIIA reduces the current amplitude without affecting the time course. Unlike DHPs, co-Aga IIIA inhibition is voltage-independent and blocks L-type channels at all potentials (Bean, 1989). However, the toxin is not specific for L-type channels as it also blocks N-type channels with high affinity. While all L-type VGCCs share the same general electrophysiological and pharmacological properties, there are differences that suggest the existence of tissue-specific forms (Bean, 1989). For example, DHP-binding affinity differs greatly depending upon the tissue type; L-type channels in smooth muscle are much more sensitive than are cardiac and skeletal muscle L-type channels and these differences cannot be entirely attributed to differences in the resting potential of the cells. L-type channels also appear to differ in a number of electrophysiological characteristics. For example, neuronal L-type channels inactivate more slowly that those in smooth muscle and cardiac cells, and the skeletal muscle channel activates significantly more slowly than native smooth and cardiac muscle L-type channels (Cognard et al, 1986). In addition, unlike L-type channels found elsewhere, the skeletal muscle channels are permeable to magnesium ions (Aimers and Palade, 1981; reviewed in Bean, 1989). L-type VGCCs play a central role in the contraction of skeletal, smooth, and cardiac muscles. The skeletal muscle L-type channel acts as a voltage sensor for excitation-contraction (E-C) coupling in skeletal muscle, presumably linking membrane depolarization to Ca 2 + release from intracellular stores. While Ca 2 + entry through this channel is not 9 required for the initiation of contraction in skeletal muscle, it may provide a source of Ca to replenish internal stores (Tanabe et al, 1988; Bean, 1989; Tsien etai, 1991; Miller, 1992). There is some evidence that L-type channels are involved in exocytotic release from endocrine cells and some neurons (Perney et al, 1986; Cazalis et al, 1987; Rane et al, 1987; Lemos and Nowycky, 1989; Wang et al, 1994) and the localization of L-type channels on the cell soma (Hell et al, 1993a) implicates these channels in the regulation of gene expression (Murphy et al, 1991; reviewed in Sutton et al, 1999). N-type channels. In addition to L- and T-type VGCCs, recordings from chick dorsal root ganglion (DRG) cells revealed a third Ca 2 + conductance of 13 pS (in 110 mM Ba2+), intermediate between that of the T- (8 pS) and L- (25 pS) type channels (Nowycky et al, 1985; Fox et al, 1987[a,b]; reviewed in Tsien et al, 1988; and Bean, 1989). Although this conductance shares some general electrophysiological characteristics with currents through both T- and L-type channels, it could not be attributed to either. Consequently, the corresponding channel was designated an N (neither)-type. Although first identified in chick DRG neurons, N-type channels have also been detected in mammalian DRG cells (Gross and MacDonald, 1987; Green and Cottrell, 1988; Petersen et al, 1989; McCarthy and TanPiengco, 1992), mammalian and amphibian sympathetic neurons (Wanke et al, 1987; Plummer et al, 1989; Jones and Marks, 1989; Carrier and Ikeda, 1992), and other cells of the peripheral and central nervous systems (Doerner et al, 1988; Williams etai, 1990; Mogul and Fox, 1991, Regan etai, 1991; Baux et al, 1992; Umemiya and Berger, 1995). N-type channels appear to be expressed only in neuronal tissues (Plummer etai, 1989; Plummer and Hess, 1991), although an N-type current has been reported in rat thyroid C-cell line (Biagi and Enyeart, 1991). Electrophysiologically, N-type channels are most easily distinguished from L-type 10 channels by their inactivation properties. Unlike L-type channels, N-type channels display time-dependent inactivation (with Ba2 + as the charge carrier). N-type currents decay with a time constant (x) ranging from 50 to 110 ms, a rate significantly slower than the rapid (x = 20 - 50 ms) inactivation of the LVA T-type channels, but much faster than the non-inactivating L-type channels. In chick DRG neurons, the N-type current decays almost completely during a test depolarization of 140 ms, while L-type current shows little inactivation over the same period of time (Nowycky et al, 1985). However, N-type currents do not always inactivate rapidly. In sympathetic neurons, the decay rate of N-type currents is much slower (x = 500 to 800 ms) and can be incomplete, even over depolarizations lasting longer than one second (Bean, 1989; Biagi and Enyeart, 1991; Plummer and Hess, 1991; Jones and Elmslie, 1992). Thus, there appears to be at least two distinct components to N-type current inactivation. These differences in inactivation kinetics could reflect different subtypes of N-type channel. Alternatively, a single N-type channel could support both currents by switching between the slow- and fast-inactivating states (Plummer and Hess, 1991; Wang et al, 1993). In addition to the time-dependence parameter, there is also a voltage-dependent aspect to N-type channel inactivation (Nowycky et al, 1985; Fox et al, 1987[a,b]; McCarthy and TanPiengco, 1992). Holding the cell membrane at potentials between -60 and -40 mV results in significant inactivation of the N-type current, and strongly negative potentials are required to reprime the channels. N-type channels are markedly more sensitive to the effects of holding potential on inactivation than are L-type channels. At resting membrane potentials of -20 mV, N-type channels are completely inactivated while L-type channels remain available for opening. Theoretically, the different inactivation properties of N- and L-type channels provides two parameters that can be used to dissect the relative contributions of the two channel types to the whole cell HVA current (Fox et al, 1987a; Bean, 1989). One method takes advantage of the different inactivation rates. The component of whole cell current that 11 decays during a prolonged depolarization can be attributed to the inactivating N-type channels, while the non-inactivating portion is identified as L-type current. The second approach exploits the different ranges over which voltage-dependent inactivation takes place. The contribution of each type to the whole cell current may be determined by analyzing the differences in whole cell currents elicited by depolarizations from resting potentials of -40 and -90 mV. Because L-type channels are relatively resistant to the effects of holding potential on inactivation while N-type channels inactivate at depolarized membrane potentials, the difference under these two conditions should reflect the contribution of N-type channels to the whole-cell current. However, neither method may be adequate to properly distinguish these currents. Some N-type channels can inactivate quite slowly and inactivation may not be complete. In addition, voltage-dependent inactivation of N-type channels can be highly variable and takes place over a wide range of holding potentials between -80 to -20 mV (Fox et al, 1987[a,b]). If N-type channels predominate in a cell, the residual current through incompletely inactivated N-type channels may be significant. Thus definitions of N- and L-type current based solely on these criteria may not be valid. N-type channels can also be differentiated from other types of VGCCs on the basis of their pharmacological profile. Likethe HVA L-type channels, N-type channels are more permeant to Ba 2 + than Ca 2 +, and are generally more sensitive to block by Cd 2 + than Ni 2 + (Nowycky et al, 1985; Fox et al, 1987[a,b]). Docherty (1988) suggested that in the presence of bicarbonate, the lanthanide gadolinium (Gd3+) selectively blocks N-type channels at concentrations between 0.5 and 5 (iM, and that T- and L-type channels are resistant to the cation. Upon exposure of rat DRG neurons to Gd 3 + in the presence of bicarbonate, there is some selective inhibition of a slowly inactivating component of whole cell Ba 2 + current suggestive of the N-type current. However, as the effect was not altered by prior exposure of the cells to co-conotoxin, Gd 3 + block does not appear to be specific to N-type VGCCs (Boland et al, 1991). In rat retinal ganglion neurons, Gd 3 + blocked all 12 HVA current while causing an increase in the LVA current (Guenther et al, 1994) and some reports indicate that Gd 3 + does not discriminate amongst VGCC types (Biagi and Enyeart, 1990; Boland et al, 1991; Canzoniero et al, 1993). In rat DRG and pituitary neurons, rat and frog peripheral neurons, and rat cardiac myocytes, Gd 3 + produced complete inhibition of all whole cell Ba 2 + currents. Gd 3 + can produce complete inhibition of VGCCs at concentrations as low as 50 nM, and other elements in the lanthanide series are also capable of producing inhibition with similar potency (Biagi and Enyeart, 1990; Boland et al, 1991). Thus, there is general agreement that, while the lanthanides are not selective for N-type channels, they are among the most potent inorganic antagonists for all VGCCs. While N- and L-type channels have similar sensitivities to the inorganic channel antagonists Cd 2 + and Gd 3 +, they can be distinguished by their sensitivities to DHPs (Nowycky et al, 1985; Fox et al, 1987[a,b]; Hirning et al, 1988; Aosaki and Kasai, 1989). In addition, N-type channels are sensitive to inhibition by a class of compounds called the co-conotoxins (Olivera et al, 1985; reviewed in Olivera et al, 1994). The co-conotoxins are a member of a family of small (13-29 amino acids) peptides found in the venom of predatory marine snails of the genus Conus. Conotoxin peptides produce paralysis by targeting different molecules in the neuromuscular system of the snail's prey. The co-conotoxins act by blocking presynaptic VGCCs, thereby inhibiting neurotransmission at the neuromuscular junction (NMJ). The first co-conotoxins identified were purified directly from the venom of three species of Conus snail: geographus, magus, and striatus. Other members of the class were later identified through molecular cloning techniques (Hillyard et al, 1992; Monje et al, 1993; reviewed in Olivera et al, 1994). All known co-conotoxins inhibit N-type VGCCs, although their specificities and blocking affinities for this particular channel vary significantly. Thus far, co-conotoxin GVIA (co-CgTx), a 27-amino acid peptide from Conus geographus (Olivera et al, 1984) is the most specific co-conotoxin peptide for N-type channel inhibition. co-CgTx produces complete and irreversible inhibition of N-type 13 currents in DRG, hippocampal, sympathetic, and sensory neurons at concentrations of approximately 100 nM to 1 u\M (Kasai et al, 1987; McCleskey et al, 1987; Aosaki and Kasai, 1989). At higher concentrations (5-15 |iM), co-CgTx also inhibits L- and T-type currents, although unlike N-type channels, the effects are incomplete and reversible (Kasai et al, 1987; McCleskey et al, 1987; Aosaki and Kasai, 1989; Wang et al, 1992; Williams et al, 1992b; Dunlap et al, 1995). In addition to co-CgTx, N-type channels are susceptible to inhibition by co-conotoxins isolated from other species of cone snail. Other distinct co-conotoxin peptides isolated from the venom of Conus magus recognize N-type channels with different specificities, co-conotoxin MVIIA produces a high affinity, albeit reversible, block of N-type channels (Yoshikami et al, 1989). co-conotoxins MVIIC and MVIID exhibit lower affinities for N-type channels and show higher affinity for the HVA DHP- and co-CgTx-insensitive subtypes of VGCC (Hillyard etai, 1992; Monje etai, 1993; Olivera etai, 1994). Radioactive and fluorescent derivatives of co-conotoxins that retain the biological activity of the native peptide have been synthesized (Olivera et al, 1994). The high affinity of co-CgTx for N-type channels (dissociation constant (Kd) ~ 50 pM), and the slow rate of dissociation once bound to the channel (10\"4 to 10\"5 per second) makes it possible to use labeled co-CgTx to study the cellular and subcellular localization of N-type VGCCs, purify channel complexes from native tissues, and study the physiological roles of N-type channels. co-CgTx binding, and by extension N-type VGCCs, is distributed throughout the PNS and CNS, including the cortex, hippocampus, olfactory bulb, and cerebellar cortex, and appear especially concentrated in regions of high synaptic density (Wagner et al, 1988; reviewed in Bean, 1989; Jones et al, 1989; Takemura et al, 1989b; Fortier et al, 1991; reviewed in Catterall et al, 1993; Wheeler et al, 1994). Although N-type channels were first identified by single channel recordings from the cell bodies of DRG neurons 14 (Nowycky et al, 1985), they appear to be more abundantly localized on dendrites and axon terminals. Single channel recordings and Ca 2 + imaging experiments also revealed N-type channels on growth cones and neurites of sympathetic neurons (Lipscombe et al, 1988). A monoclonal antibody specific to co-CgTx revealed immunoreactivity at the branch points of Purkinje cell dendrites (Fortier et al, 1991) and a number of studies have used fluorescently-labeled or biotinylated co-CgTx to demonstrate the presence of N-type VGCCs at the NMJ (Robitaille et al, 1990; Cohen et al, 1991; Tarelli et al, 1991; Farinas et al, 1993). co-CgTx binding occurs at the active zones of the presynaptic cell, in spatial register with the postsynaptic acetylcholine (ACh) receptors on the muscle. Labeling is rarely found between active zones, nor is it localized to areas of the presynaptic membrane that do not face the muscle. N-type channels have also been observed to cluster in areas of synaptic contact on hippocampal CA1 neurons (Jones et al, 1989). The subcellular localization of N-type channels is highly suggestive of their physiological roles. Their presence on the presynaptic membrane suggests that Ca 2 + entry through these channels is responsible for triggering neurotransmitter release. An early study (Kerr and Yoshikami, 1984) demonstrated that co-CgTx blocks electrically-induced release from the frog NMJ and numerous studies have demonstrated that the application of co-CgTx inhibits neurotransmitter release in the central and peripheral nervous system (Dooley et al, 1988; Lundy and Frew, 1988; Dutar et al, 1989; Herdon and Nahorski, 1989; Takemura et al, 1989a; Wessler et al, 1990; Home and Kemp, 1991; Potier et al, 1993). Furthermore, biochemical studies indicate that N-type channels are physically associated with proteins such as synaptotagmin and syntaxin which are part of the exocytotic machinery (Leveque et al, 1992; Leveque et al, 1994; Sheng et al, 1994). There are species- and cell-specific differences in N-type-channel-regulated neurotransmission. While co-CgTx completely abolishes neurotransmission at the avian and amphibian NMJ, it has no effect on the mammalian motor nervous system (Kerr and Yoshikami, 1984; Sano etal, 1987; Wessler etal, 1990; Protti etal, 1991; reviewed in 15 Adams and Olivera, 1994). The ability of this toxin to inhibit neurotransmission also varies depending on the type of synapse within a given species (Wessler et al, 1990; Home and Kemp, 1991; Potier et al, 1993; reviewed in Dunlap et al, 1995). For example, inhibitory synaptic transmission in hippocampal CA1 neurons is strongly reduced by the application of co-CgTx, whereas the toxin blocks excitatory transmissions to a much lesser extent. In addition, while co-CgTx inhibits release of ACh from both autonomic and central neurons in the rat, release from central neurons is approximately 20-fold less sensitive (Wessler et al, 1990). In spite of the complete and irreversible inhibition of N-type channels produced by co-CgTx, application of the toxin to many types of neurons only partially inhibits neurotransmitter release, suggesting that other types of VGCCs contribute to neurotransmitter release from both central and peripheral neurons (Wheeler et al, 1994; Wu and Saggau, 1994; Turner et al, 1995). In fact, while regulation of transmitter release from peripheral neurons appears to predominantly involve N-type channels, release in the central nervous system appears to be controlled primarily by other types of VGCCs that are insensitive to both co-CgTx and DHPs (Burke et al, 1993; Luebke et al, 1993). The presence of N-type VGCCs in regions other than the synapse indicates that these channels have other functions in addition to neurotransmitter release. N-type channels localized to dendritic branch points may be involved in integration or amplification of neural inputs (Fortier et al, 1991). N-type channels may also play a role in nervous system development as evidenced by the expression of N-type channels on postmitotic cerebellar granule cells. These cells only begin migration after the appearance of N-type channels and co-CgTx causes a cessation of migration (Komuro and Rakic, 1992). 16-Other HVA VGCCs: P-, Q-, and O-type channels. The original classification system of VGCCs, which was expanded from the simple LVA/ HVA dichotomy to encompass T-, L- and N-channels, was subsequently found to be too restrictive to adequately describe all VGCCs. The availability of blocking agents that target L- and N-type channels revealed other HVA currents that could not be defined according to this scheme (Bean, 1991; Mogul and Fox, 1991; Randall and Raabe, 1992; Regan, 1991; Regan et al, 1991; reviewed in Olivera et al., 1994). Additionally, the partial inhibition of transmitter release produced by co-CgTx at some synapses and its complete failure to block neurotransmission at other synapses such as the mammalian NMJ suggested the presence of a novel channel type that also mediates neurotransmission (Seabrook and Adams, 1989; Wessler et al., 1990; Protti et al., 1991). These novel channel types, variously named P-, Q-, O-, and R-, have primarily been defined on the basis of their distinctive pharmacological properties rather than electrophysiological characteristics. The P-type current was originally identified as an HVA current in Purkinje cells that is insensitive to the agents typically used to inhibit L- and N-type channels (Llinas et al., 1989). These channels are thought to support the Ca2+-dependent action potentials in the dendrites of cerebellar Purkinje cells, which are unaffected by DHPs and co-CgTx, but are potently blocked by components of the venom of the funnel web spider Agelenopsis aperta (Llinas etal, 1989; Lin etal, 1990; Mintz etal, 1992a; Bindokas etal, 1993). Whole cell recordings from Purkinje cells reveal an HVA current that peaks at voltages between -30 and -20 mV and inactivates slowly over the duration of the depolarization (Regan, 1991; Regan etal, 1991; Mintz etal, 1992b; reviewed in Dunlap et al, 1995). Single channel analysis of P-type channels reveals conductances in ranges similar to those of N- and L-type channels. Multiple unitary conductance levels of 9, 14, 19 pS in 110 mM Ba 2 + have been reported for P-type channels in the Purkinje cell soma and dendrites (Usowicz et al, 1992), and a P-type current in hypoglossal motomeurons 17 has a unitary conductance of 20 pS (Umemiya and Berger, 1995). P-type channels purified from cerebellar tissue and reconstituted in lipid membranes displayed single channel conductance levels of 10 -12 pS in 80 mM Ba 2 + and 5 - 8 pS in 100 mM Ca 2 + (Llinas etai, 1989). The venom of the funnel web spider, like that of the cone snail, is a cocktail of toxins that target different elements of the synaptic machinery. FTX, a non-peptide component of the venom (arginine polyamine and a synthetic analog of FTX, sFTX), were initially reported to be specific blockers of P-type channels (Llinas et al, 1989; Lin et al, 1990; reviewed in Scott etai, 1991), but were subsequently shown to produce inhibition of other Ca 2 + currents in conjunction with the P-type block (reviewed in Scott et al, 1991; Olivera etai, 1994). co-Aga IVA, a 48-amino acid peptide also found in the venom of A. aperta potently inhibits both voltage-dependent Ca 2 + entry into rat brain synaptosomes and the HVA current in Purkinje cells, as well as a portion of the HVA current in a variety of other neurons (Mintz et al, 1992[a,b]; reviewed in Dunlap et al, 1995). At concentrations of 100 to 200 nM, co-Aga IVA has no effect on T-, L-, and N- type currents. In Purkinje cells, complete inhibition is observed at concentrations below 200 nM, with half-maximal block produced at concentrations between 2 and 10 nM. Inhibition is rapid, occurring within two minutes of application, and while the inhibition is poorly reversible by wash-out, it can be removed by a series of strong depolarizations (i.e. to +70 mV). Block of P-type currents by co-Aga IVA in other neurons is qualitatively similar to that in Purkinje cells although the kinetics vary slightly. For example, while inhibition of P-type current in spinal cord interneurons and neurons in the visual cortex occurs as rapidly as that in the Purkinje cells, the rate of block is several times slower in CA1 and CA3 hippocampal neurons (Mintz etai, 1992b). A peptide toxin isolated from the cone snail Conus magus has also been shown to inhibit P-type VGCCs (Hillyard et al, 1992). This toxin, co-CTx MVIIC, blocks P-type 18 channels with an IC 5 0 of 1 - 10 LiM. However, co-CTx MVIIC also inhibits N-type channels as well as the HVA Q- and O-type VGCCs (see below, Hillyard et al, 1992). P-type channels are especially prominent in, but not restricted to, Purkinje cells. Xenopus oocytes injected with mRNA isolated from rat brain express an co-CgTx and DHP-insensitive current that is blocked by FTX, and HVA currents have been recorded from rat DRG and motorneurons that are sensitive to block by co-Aga IVA, suggesting that the P-type channel is widespread in the mammalian central and peripheral nervous system (Lin etal., 1990; Regan, 1991; Regan etal, 1991; Mintz etal, 1992[a,b]; Usowicz etal, 1992; Umemiya and Berger, 1995). At saturating concentrations of co-Aga VIA (60 - 200 nM), 85 - 100% of the HVA current in Purkinje cells is blocked (Mintz et al, 1992[a,b]). Furthermore, 85 - 100% of the HVA current in Purkinje cells remains after combined application of co-CgTX and nitrendipine (Regan, 1991; Regan etal, 1991). These results indicate that the P-type channel is the primary source of HVA current in these neurons. However, while all non-L-, non-N-type current in Purkinje cells appears to be carried by P-type channels, this is not true for other cells in the cerebellum and other parts of the central and peripheral nervous system (Mintz etal, 1992[a,b]; Regan, 1991; Regan etal, 1991). In spinal cord neurons, hippocampal neurons, and cells in the visual cortex, co-Aga IVA blocks a much smaller percentage of HVA current, and sympathetic neurons appear to lack co-Aga IVA sensitive currents (Mintz et al, 1992b). P-type channels do not account for all of the DHP- and co-CgTx-resistant current in neurons since a substantial fraction of current remains even after exposure to saturating concentrations of DHPs, co-CgTx, and co-Aga IVA. Cultured rat cerebellar granule cells express an HVA current that is unaffected by these inhibitors at concentrations which block L-, N- and P-type channels, respectively (Zhang etal, 1993; Olivera etal, 1994; Randall and Tsien, 1995). However, the channels supporting this novel current (termed Q-type) are partially blocked by co-Aga IVA at concentrations 10 to 100 times that required for P-type inhibition and are completely blocked by co-CTx MVIIC (IC50= 30 to 300 nM). In 19 addition to differing sensitivities to these toxins, Q-type channels partially recover from co-Aga IVA-induced inhibition within minutes of toxin washout. Q-type channels also display electrophysiological properties distinct from those of P-type channels. While P-type currents in Purkinje cells and cerebellar granule cells show no inactivation over a 100 ms test depolarization, the Q-type current in granule cells decays to approximately 65% of the peak current over the same time period. The existence of O-type channels has been inferred solely from pharmacological studies. O-type channels were identified as high affinity co-CTx MVIJC binding sites (Adams et al., 1993). These channels are significantly more sensitive to the toxin than are other co-CTx MVUC-sensitive channels (reviewed in Olivera et al., 1994). It is possible that P-, Q-, and O-type channels are members of the same channel family which possess slightly different pharmacological and electrophysiological properties as a result of alternative splicing of the a, subunit gene and/or different complements of auxiliary subunits. The cellular and subcellular distribution of P-type channels in the mammalian nervous system has been studied using polyclonal antibodies against purified P-type channels (Hillman et al, 1991). Staining is most intense on the Purkinje cell dendrites, particularly at the bifurcation points. Less intense labeling was observed on cell bodies and proximal dendrites of the Purkinje cells, and no immunoreactivity was present in cerebellar granule cells. P-type channels are also present on the dendrites and cell bodies of neurons throughout the CNS, including the olfactory bulb, brainstem, frontal cortex, and hippocampus. An anti-peptide antibody generated against a cloned a, subunit believed to give rise to both P- and Q-type channels (a1A, see below) shows a similar cellular distribution pattern (Westenbroek etai., 1995), as do in situ localization studies using antisense DNA and RNA probes to the a 1 A (Stea et al., 1994). The a 1 A probes also recognized regions which were left unlabeled by the polyclonal antibody generated by 20 Hillman etal. (1991), presumably reflecting the localization of both Q-type and P-type channels. While O-type channels have not been localized immunohistochemically, evidence from binding studies suggests that they are widely distributed in the mammalian CNS (Olivera et al., 1994). Estimates of O-type bindings sites in rat brain preparations suggest that O-type channels are more prevalent than N-type channels in the CNS, and it has been proposed that O-type channels are localized exclusively to synaptic termini, which would largely prevent their detection through electrophysiological means. Many studies implicate P-, Q, and O-type channels in neurotransmitter release (reviewed in Olivera et al, 1994). While N-type channels mediate release at some synapses in the mammalian CNS, the co-Aga IVA-sensitive channels appear to play a more prominent role (Luebke et al, 1993; reviewed in Dunlap et al., 1995). co-Aga IVA potently blocks Ca 2 + uptake into synaptosomes (Mintz et al, 1992a) and partially inhibits the release of dopamine and glutamate from synaptosomes (Turner et al, 1992; Turner et al, 1993) and at CA1-CA3 synapses in the hippocampus (Burke etal, 1993; Wu and Saggau, 1994; Wheeler et al, 1994). In the peripheral nervous system, co-Aga IVA has little or no effect on the autonomic nervous system (Lundy and Frew, 1994), but P-type channels are probably responsible for neurotransmitter release at the mammalian NMJ (Uchitel et al, 1992; Bowersox et al, 1993). As co-Aga IVA blocks both P- and Q-type channels, it is possible that both channel types are involved in neurotransmission. Wheeler and colleagues found that the pharmacological properties of the co-Aga IVA-sensitive channels supporting neurotransmission in the hippocampus and for co-Aga IVA-induced block of inhibitory postsynaptic potentials in the cerebellum were more similar to Q- than P-type channels (Takahashi and Momiyama, 1993; Wheeler et al, 1994). Finally, O-type channels may also mediate neurotransmission at certain synapses, as norepinephrine release in the hippocampus is inhibited by subnanomolar concentrations of co-CTx MVLIC (reviewed in Olivera etal, 1994). 21 R-type current. A component of the HVA current in cerebellar granule cells remains even after the application of nimodipine, co-CgTx , co-Aga VIA, and co-CTx MVIIC. This current, categorized as R (residual or resistant)- type (Zhang et al, 1993), comprises approximately 15% of the HVA current in these cells. R-type current may not necessarily reflect a single channel type, but a family of molecularly distinct channels with similar pharmacological and electrophysiological characteristics. R-type current begins to activate around -40 mV and reaches a peak amplitude at 0 mV. The current inactivates rapidly, and the increased rate of inactivation with Ca 2 + as the charge carrier suggests that the channels supporting the R-type current inactivate in a Ca 2 +-dependent manner. R-type channels are equally sensitive to block by Cd 2 + and N i 2 + ions. The exact nature of the channels supporting this current is currently unknown. See the section on Class E for further discussion. HVA Ca 2 + Channels are Multiprotein Complexes. Biochemical studies have established that VGCCs are multiprotein complexes. By taking advantage of the high-affinity binding of organic VGCC antagonists, several groups have purified the L-type channel from skeletal muscle. Four distinct polypeptides, designated a, (175-kDa), cx28 (170-kDa), (3 (52-kDa), and y (32-kDa), comigrate with the ligand-binding activity (Curtis and Catterall, 1983; Flockerzi etai, 1986; Takahashi etai, 1987; reviewed in De Waard et al, 1996). A minor 212-kDa band also co-purifies and has been shown to represent a larger, much less abundant form of the skeletal muscle a, subunit (De Jongh et al, 1989). Similar approaches have been used to isolate the cardiac L-type (Norman and Leech, 1994) and brain N-type channels (Witcher et al, 1993). These complexes also 22 consist of an a, subunit associated with (3 and a28 subunits. The P and a28 are highly similar, if not identical to, the subunits associated with the skeletal muscle a, (Ahlijanian et al, 1991; Sakamoto and Campbell, 1991). However, unlike the skeletal muscle L-type channel, no y subunits appeared as part of either complex. A novel 95-kDa polypeptide was found to comigrate with the N-type channel although it is unclear whether this represents a bonafide channel subunit or merely a proteolytic fragment (Witcher et al, 1993). While subunit composition differs slightly depending on channel type, a general model has been proposed in which four to five proteins form a multisubunit complex (Figure 1 A). In this model, the ax subunit forms the channel proper, comprising both the voltage-sensing mechanism and the Ca 2 + selective pore, and the remaining proteins interact with the a, subunit to modulate its activity. Structure and properties of VGCC a,, subunits. The first cDNAs encoding VGCC subunits were isolated from rabbit skeletal muscle (designated a l s according to the current nomenclature) (Tanabe et al, 1987; Ellis et al, 1988). The a i s is an 1873-residue protein that bears a high degree of amino acid similarity to the voltage-gated Na+ and potassium (K+) channels. The a, subunit is predicted to consist of four homologous, mainly hydrophobic domains (designated domains I, II, III and IV). Each of the four domains is comprised of six putative membrane-spanning segments (S1-S6). The S4 segment in each domain contains positively-charged residues every third or fourth position and is believed to form part of the voltage-sensing mechanism of the channel. Between the S5 and S6 segments of each domain are two hydrophobic segments, SSI and SS2, which are predicted to form the channel pore (Figure IB). Based on similarity to the voltage-gated Na+ channel, Tanabe et al. (1987) speculated that the VGCC a, subunit may form both the Ca2+-selective pore and the voltage 23 Figure 1. Composition of a VGCC complex and structure of the 0Cj subunit. A) Diagram of a VGCC complex, indicating the a,, a2/8, (3, and y subunits. The a, subunit forms the channel proper, comprising both the voltage-sensing mechanism and the Ca2+ selective pore, and the remaining proteins are thought to interact with the a, subunit and modulate its activity. B ) Predicted structure and transmembrane topology of the a , subunit. The pore-forming SS1-SS2 loops are shown in bold. The conserved EF hand motif in the carboxyl terminus, the (3-subunit binding site in the I-II linker, the G-protein |3y-subunit binding sites in the I-II linker and carboxyl terminus, and the regions responsible for functional interaction with the synaptic release machinery ( a 1 A , a ] B ) and the ryanodine receptor (ais) are indicated. 24 25 sensor of the channel complex. This hypothesis was supported by studies demonstrating that expression of the a l s in myotubes from dysgenic mice restored normal skeletal muscle-type E-C coupling and the slow Ca 2 + current absent in these cells (Tanabe et al, 1988). In addition, a i s expression in dysgenic myotubes restored the charge movement observed in normal myotubes upon membrane depolarization (Adams etal, 1990). These results indicated that the skeletal muscle a i s subunit acts both as a voltage-sensor, providing a physical connection between membrane depolarization and Ca2+-release from intracellular stores for the initiation of muscle contraction, and is also part of a functional VGCC. Using the skeletal muscle clone as a probe, cDNAs encoding homologous a, subunits have been subsequently cloned from cardiac (Mikami etal, 1989) and smooth (Biel et al., 1990; Koch et al., 1990) muscle. Injection of the cardiac a, subunit into dysgenic myotubes resulted in the expression of a VGCC which differed markedly in terms of activation rate, Ba 2 + permeability, and E-C coupling from the current conducted through channels formed by the skeletal muscle clone (Tanabe et al, 1990b). Expression of cardiac and smooth muscle a, subunit clones' in Xenopus oocytes (Mikami et al, 1989; Biel et al, 1990; Bosse et al, 1992; Itagaki et al, 1992) resulted in large inward currents that were sensitive to the organic channel agonists and antagonists, thereby identifying them as L-type channels. Co-expression of skeletal muscle-derived ofi and [3 subunits, while affecting the amplitude and voltage dependence of the currents, was not required for channel activity or drug binding, suggesting that the a, subunit is capable of forming a functional channel in the absence of the other subunits. However, because some VGCC subunits may be endogenously expressed by Xenopus oocytes (Singer-Lahat et al, 1992; Tareilus et al, 1997), it is possible that the a, protein forms a complex with these endogenous auxiliary subunits. This prompted several groups to examine the properties of the a, subunit in cells lacking these proteins. Murine L-cells (Perez-Reyes et al, 1989; Kim et al, 1990) and Chinese Hamster Ovary (CHO) cells (Bosse et al, 1992) stably transformed with a, subunits express voltage activated Ca 2 + currents sensitive to L-type 26 channel blockers. While Ca + currents in cells expressing the smooth muscle a, subunit displayed similar drug sensitivities and kinetics to the native currents, the currents supported by a i s activated considerably more slowly than currents recorded from skeletal muscle cells. At least nine different a, subunit genes are now known to be expressed in the mammalian nervous system. Initially, several a, subunit cDNAs were isolated from a rat brain library on the basis of their homology to the rabbit skeletal muscle a, subunit (Snutch et al, 1990). Each cDNA hybridized to one of four distinct banding patterns on Northern blots of rat brain mRNA, allowing them to be grouped into four classes, designated A, B, C, and D. Subsequently, a fifth o^ subunit (class E) was isolated from rat brain (Soong et al, 1993). Southern blot analysis and DNA sequencing indicated that these five classes are separate members of a multigene family. Homology analysis indicated that the class A, B, and E channels are more similar to one another than they are to the class C and D channels (Figure 2). The class C clone is almost identical to the cardiac a, subunit, suggesting that the class C and D clones represent members of the DHP-sensitive L-type channels, while the A, B, and E clones are DHP resistant. Four other VGCC a, subunit genes have been identified in the mammalian genome. The oc1F (Strom et al, 1998) shares the most sequence identity with the L-type channels. The oc1G, a 1 H, and ocn clones represent the LVA branch of the VGCC family (Perez-Reyes etai, 1998; Cribbs etai, 1998; J.-H. Lee etai, 1999). The individual a, subunit clones share the most homology in the transmembrane domains with the majority of sequence divergence occurring in the putative cytoplasmic regions of the channels. The loop between domains II and III, and the cytoplasmic tail vary in size as well as sequence. The DHP-sensitive channels (classes C, D, F, and S) have relatively short (=130 amino acid) sequences linking domains II and III while the analogous region in the class A and B channels are significantly larger (~ 430 amino acid). However, despite the size similarity between the linkers, the class A and B clones show 27 Figure 2. Similarity tree of mammalian VGCC a, subunits. The predicted amino acid sequences of representatives of each class of VGCC a, subunit and Na+ channel a subunits were compared pairwise and the percent similarities were plotted. GenBank Accession Numbers for VGCCs: rat a1A, M64373; rat a1B, M92905; rat a l c , M67515; rat cc1E, L15453; human a 1 F , AJ224874; rabbit cxis, M23919; rat 1 x 10\"3) were established and maintained by picking single Unc animals to new plates. Revertants were identified on the basis of their improved movement compared with their Unc siblings. Revertants were maintained for several generations until homozygous strains were established, and then PCR was used to amplify the region surrounding the site of excision. The PCR products were then sequenced by PCR cycle sequencing. Phenotypic analysis of unc-2 mutants. Aldicarb and Nicotine Resistance. NGM plates were prepared according to the standard method. Aldicarb (Sigma) was prepared as a 105 mM stock solution (lg/50 ml) in ethanol and added to the agar growth medium to a final concentration of 0.5 mM after autoclaving (Miller et al, 1996). After seeding with OP50, plates were stored in the dark at 4\u00C2\u00B0. Individual animals were placed on plates and incubated at 20\u00C2\u00B0. Animals were observed after lh, and 1, 2, and 3 weeks. To test for sensitivity to nicotine, plates were flooded with 1% nicotine solution. Thrashing Assay. Young adult hermaphrodites were transferred to a microtiter well containing 60 \x\ of M9 buffer at ambient room temperature, - 2 0 - 2 3 \u00C2\u00B0 . After a 2 min. recovery period, thrashes were counted for 2 min. (Miller et ai, 1996). A thrash was defined as a change in direction of bending at the mid-body. Ten animals from each strain were examined. 74 Expulsion Assay. Young adult hermaphrodites were examined with a dissecting microscope for the presence or absence of an expulsion event after each posterior body muscle contraction (pBoc) of the defecation cycle (Miller et al., 1996; Avery and Thomas, 1997). Ten animals from each strain were observed for 10 consecutive cycles. The assay was carried out at ambient room temperature, 20-23\u00C2\u00B0 . Molecular biology. Standard molecular biology techniques were used (Sambrook et al., 1989). Plasmid subcloning was done using the pBluescript KS- (Stratagene), pucl8 (Gibco-BRL), pSL (Pharmacia), pCR2.1 (Invitrogen), pGEM-T (Promega), pGEM-T Easy (Promega), and pGEX vectors. Ligation reactions were transformed into either XL-II electrocompetent cells or DH5a chemically competent cells. Bacterial colonies were grown on 2x YT plates containing 15 g agar/1 and antibiotic at the appropriate concentration. Whenever possible, colonies carrying recombinant plasmids were identified through blue-white selection. All PCR reagents were supplied by Gibco-BRL, unless otherwise specified. All primers mentioned in the text are listed in Table 4. DNA sequence of constructs was determined by the dideoxy method using the Sequenase 2.0 kit (U. S. Biochemical Corporation). DNA sequences of fragments amplified from C. elegans genomic DNA were determined by cycle sequencing of the PCR products (see below). PCR amplification procedures. RT-PCR. Reverse transcription-PCR was performed using two different methods. In both methods, single-stranded cDNA for PCR amplification was prepared by annealing 25 pmol 75 downstream primer, random hexamer (BRL), or oligo dT (BRL) primer to ~ 1.0 JLLI total RNA. In Method #1, a cocktail consisting of 2.0 ul MgCl2 (25 mM), 1.0 pi PCR buffer (lOx), 4.0 pi dNTP mix (2.5 mM each), 0.5 pi RNasin, and 0.5 pi Superscript II reverse transcriptase (Gibco-BRL) was added, and the reaction was incubated at 42\u00C2\u00B0 for lh, followed by 99\u00C2\u00B0 for 15 min. Reactions were then heated to 70\u00C2\u00B0 for 5 min. before adding 38 pi of a cocktail consisting of 4.0 ul MgCl2 (25 mM), 4.0 pi PCR (lOx), 3 pi DMSO, 26.75 jLXl dH 20, and 0.25 pi Taq DNA Polymerase (Gibco-BRL), and 1.0 JLLI each of up-and downstream primers at 25 pmol/pl. In Method #2, 8 ul of a master mix containing 1.0 pi PCR buffer (lOx), 1 pi dNTP mix (2.5 mM each), 1 pi 100 mM DTT, 1.0 pi MgCl2 (25 mM), 0.5 pi RNasin, and 0.5 pi Superscript II reverse transcriptase (Gibco-BRL) was added to the annealed RNA. The reaction was incubated at 42\u00C2\u00B0 for lh, followed by 99\u00C2\u00B0 for 15 min. Several drops of mineral oil were added to each reaction to prevent evaporation and a \"hot start\" was performed in which the reactions were heated to 70\u00C2\u00B0 for 5 min. and a cocktail containing 1.5 pi PCR buffer, 1.0 pi dNTP mix (2.5 mM each), 1.0 pi each forward and reverse primer (25 pmol/pl), and dH 20 to 25 pi was then added. The mixture was then amplified for 25-30 cycles consisting of 1 min. at 95\u00C2\u00B0, 30 sec. at x, and y sec. at 72\u00C2\u00B0, followed by a 5 min. incubation at 72\u00C2\u00B0, where x = 5\u00C2\u00B0 below the predicted melting temperature of the oligonucleotide and y = 60 sec/kb of amplification product. PCR. Amplification from a double-stranded DNA template was performed in an similar manner, except that the template was incubated at 70\u00C2\u00B0 for 5 min. in a volume of 10 pi under mineral oil. After the hot start, 15 pi of a cocktail consisting of 2.5 pi lOx buffer, 2.0 pi dNTPs (2.5 mM each), 1.0 pi MgCl2 (25 mM), 1 pi forward primer (25 pmol), 1 pi reverse primer (25 pmol), and 0.5 pi Taq polymerase (Gibco-BRL), and dH20 to a final volume of 25 pi. 76 5' R A C E . 5' RACE was performed according to the procedure outlined in the 5' RACE System (Gibco-BRL). All reagents (except EM primers) were included in the kit unless otherwise noted. Reverse transcription of RNA from wild-type adult hermaphrodites was performed. 1.0 ixg of RNA was annealed with 2.5 pmoles of an antisense primer. A reaction mixture consisting of 2.0 |ll 25 mM MgCl2 (Gibco-BRL), 2.0 |ll lOx PCR buffer (Gibco-BRL), 4.0 |il 2.5 mM each dNTP (Gibco-BRL), 0.5 ul RNase inhibitor (RNasin; Gibco-BRL), and 0.5 \i\ reverse transcriptase (RTase U; Gibco-BRL) was added and samples were incubated for lh at 42\u00C2\u00B0. Following a 15 min. incubation at 70\u00C2\u00B0 to inactivate the enzymes, 1 |0,1 RNase Mix was added, and the mixture was incubated at 37\u00C2\u00B0 for 30 min. The cDNA was then purified using a GlassMax spin cartridge according to the procedure outlined in the manual. A poly(C) tail was added by combining 5.0 (xl 5x tailing buffer, 2.5 |il 2.0 mM dCTP, 6.5 \ i \ dH20, and 10 (J.1 of the prepared cDNA. The mixture was heated to 95\u00C2\u00B0 for 3 min. to eliminate secondary structure, 1 |il terminal transferase was added, and the reaction was incubated at 37\u00C2\u00B0 for 10 min. followed by 65\u00C2\u00B0 for 10 min. 2.5 uj of poly(C) cDNA was amplified with 2.0 |ll primer AAP (1.25 pm/u\l) and 2.5 pmol of a nested antisense primer for 29 cycles consisting of 4 cycles of 30 sec. at 95\u00C2\u00B0, 30 sec. at 49\u00C2\u00B0, 1 min. at 72\u00C2\u00B0, a second set of 4 cycles of 30 sec. at 95\u00C2\u00B0, 30 sec. at 35\u00C2\u00B0, 1 min. at 72\u00C2\u00B0, and 25 cycles of 30 sec. at 95\u00C2\u00B0, 30 sec. at 49\u00C2\u00B0, 1 min. at 72\u00C2\u00B0. A second round of amplification was performed on 1.0 (ll of the PCR product using primers AUAP and the nested antisense primer. 25 cycles of 30 sec. at 95\u00C2\u00B0, 30 sec. at 49\u00C2\u00B0, and 1 min. at 72\u00C2\u00B0 was performed. 1.0 (il of the second round amplification product was subcloned into a plasmid vector. 77 Genomic DNA Preparation and Amplification. Genomic DNA was amplified as described by Barstead et al. (1991), with some modifications. In most experiments, worms were washed from crowded 6 cm plates with M9 buffer or distilled water. The animals were pelleted by centrifugation and the supernatant carefully aspirated without disturbing the pellet. The pellet was washed with M9 or water to remove residual bacterial contamination. After re-centrifugation and aspiration of the supernatant, the pellet was transferred to an 0.5 ml microfuge tube containing 250 pi of worm lysis buffer (50 mM KC1, 10 mM Tris (pH 8.0), 2.5 mM MgCl2, 0.45% Tween 20, 0.45% NP-40, 60 mg/ml proteinase K). The pellet was gently dispersed throughout the lysis buffer by gentle agitation, transferred to fresh 0.5 ml tubes in 50-100 pi aliquots, and overlaid with mineral oil to prevent evaporation. The tubes were incubated for 45 min. at 60\u00C2\u00B0, followed by a 15 min. incubation at 95\u00C2\u00B0 to inactivate the Proteinase K. Debris was pelleted by centrifugation and the DNA-containing supernatant was transferred to a fresh tube and stored at -20\u00C2\u00B0. Five microliters of the crude DNA preparation used in each 25 pi PCR reaction volume, and amplification was performed as described above. RNase Protection Assay. The RNase Protection Assay was performed according to the procedure outlined in the Mismatch Detect LI kit (Ambion) with some modifications. Primer pairs were designed such that the entire unc-2 genomic sequence could be amplified using the PCR in segments of approximately 800 to 1000-bp. Nested primers were designed for each segment. The T7 RNA polymerase consensus sequence was incorporated into the upstream nested oligonucleotide at the 5' end. Similarly, the SP6 RNA polymerase consensus sequence was incorporated onto the 5' end of the downstream nested primer (Figure 3). 78 Figure 3. Diagram of the RNase Protection Assay. A) Fragments of unc-2 genomic sequence are amplified using PCR. The fragments are re-amplified using nested primers to incorporate T7 and SP6 RNA polymerase recognition sequences. B) Each fragment is transcribed with T7 or SP6 RNA polymerase to generate single-stranded RNA. C) T7 transcripts from mutants are hybridized to SP6 transcripts from wild-type animals and vice versa. D) Double-stranded RNA is digested with RNase. E) The digested RNA is analyzed on an agarose gel and visualized with Ethidium Bromide. 79 unc-2(ra611) Wild-type unc-2(ra612) 80 Genomic DNA from unc-2 mutants and N2 animals was prepared and amplified using the methods described above. Initial PCR reactions were performed using the primer pairs to generate a smaller template, facilitating the second round of PCR. Second round amplification using internal primers was performed using 1 to 5 pi of the PCR product obtained in the first amplification. The DNA was then amplified for 4 cycles consisting of 30 sec. at 95\u00C2\u00B0, 30 sec. at x\u00C2\u00B0, and y sec. at 72\u00C2\u00B0, followed by 20 cycles consisting of 30 sec. at 95\u00C2\u00B0, 30 sec. at 72\u00C2\u00B0, and y sec. at 72\u00C2\u00B0 and finally a 5 min. incubation at 72\u00C2\u00B0. This round incorporates the RNA polymerase recognition sites onto the PCR product. Single-stranded RNA was transcribed using the second-round amplification product as the template. To generate the T7 transcript, 2 JLXI of template DNA was combined with 1 pi lOx transcription buffer (Ambion), 2 pi rNTPs (Ambion), 1 pi T7 RNA polymerase (BRL), and 4 (Xl dH20. The mixture was incubated at 37\u00C2\u00B0 for one hour. The SP6 transcript was produced by combining 2 (ll template, 2 pi 5x SP6 transcription buffer (BRL), 2 |ll rNTPs (Ambion), 1 pi 10 mM dithiothreitol (BRL), 1 pi SP6 RNA polymerase (BRL), and 2 LLI dH20 and incubating at 42\u00C2\u00B0 for one hour. The 10 pi reaction volume provides sufficient quantities of the mutant transcripts. The reactions generating the N2 transcripts were scaled up to provide enough RNA to hybridize to each of the mutant transcripts. After the incubation period, 2 |il samples of the single stranded RNA were analyzed on a 2.0% agarose gel containing 0.1 P-g/ml ethidium bromide to assess the amount of T7 mutant transcript relative to the N2 SP6 transcript and vice versa. An equal volume (8 pi) of Hybridization Buffer (Ambion) was added to each of the remaining samples. T7 transcripts from mutant animals were combined with an equal amount of N2 SP6 transcript while the SP6 mutant transcripts were mixed with an equal amount of T7 transcript. Finally, to provide a control, N2 T7 transcripts were combined with N2 SP6 transcripts. Any secondary structure present in the RNA was eliminated by heating the samples to 95\u00C2\u00B0 81 for 3 min. The samples were allowed to cool slowly to room temperature to anneal the strands of RNA. Four microliter aliquots of the double-stranded RNA were digested with the three RNase mixtures provided by Ambion. RNA was added to 16 pi of Digestion Buffer (Ambion) and 1 pi of either RNase Mix 1, 2, or 3, and incubated at 37\u00C2\u00B0 for 45 min. Four microliters of Loading Dye (Ambion) was added to the digestion reactions and the entire volume was run on a 2.0% agarose (BRL) gel. Samples were deemed positive for a mutation if there was a shift in the molecular weight of the RNA band compared with the control band. The approximate position of the mutation could be determined by the magnitude of the shift. Cycle Sequencing of PCR Products. Once a mutation had been localized to a specific region, the PCR product was sequenced directly using the BRL dsDNA Cycle Sequencing System. Sequencing reactions consisted of 1 - 5 pi of the PCR product derived from the first round of amplification, 4.5 pi lOx sequencing buffer, 0.5 pi Taq polymerase, 5 pi 32P-labeled primer (1 pmol), and dH20 to a final volume of 36 pi. Two microliters of the appropriate termination mix and 8 pi of the sequencing reaction mix were added to each of four tubes labeled A, C, G, and T. A drop of mineral oil was added to each tube and contents were amplified for 20 cycles consisting of 30 sec. at 95\u00C2\u00B0, 30 sec. at x\u00C2\u00B0, and 1 min. at 72\u00C2\u00B0, followed by 10 cycles consisting 30 sec. at 95\u00C2\u00B0 and 1 min. at 72\u00C2\u00B0. After amplification, 5 pi of stop solution was added to each tube and reactions were heated to 95\u00C2\u00B0 for 5 min. before loading on a 6% poly acrylamide sequencing gel. 82 Screening of the AACT-RB2 cDNA library. unc-2 sequences were isolated from the random hexamer-primed C. elegans cDNA library AACT-RB2 obtained from R. Barstead. The RB3E and RB4E bacterial strains were provided with the phage stock. NZYCM growth media containing (in g/1 of media): NZ Amine, 10; NaCl, 5; Casamino acids, 1; Yeast extract, 5; MgS04, 2, A, dil contained (ml/100 ml): IM Tris pH 7.5, 1; IM MgS04, 2. Culture plates consisted of NZYCM plus 14 g/1 of agar. Top agarose consisted of NZYCM media containing 2% agarose. Three rounds of plaque purification were performed; the first round was carried out on 150 x 15 mm culture plates. The second and third rounds were carried out on 100 x 15 mm plates. The probe was generated by amplifying a 240-bp fragment from the AACT-RB2 library with the primer set EM82-EM83. The PCR product was subcloned into pBluescript and the sequenced determined. A 187-bp fragment was excised with EcoJU and HindlJl and gel purified. 25 ng of the purified fragment was labeled with [a-32P]dATP and [oc-32P]dCTP according to the procedure outlined in the Random Priming kit (BRL). Fifteen 150 x 15 mm NZYCM plates were screened in the first round of plaque purification. A single colony of RB3E was used to inoculate a 10 ml culture of NZYCM media containing 0.2% maltose and incubated overnight at 37\u00C2\u00B0. The RB3E culture was gently pelletted and resuspended in 5 ml X dil. The phage stock solution was diluted 10,000-fold in A, dil. Fifteen Falcon tubes containing 20 pi of diluted phage and 200 pi of RB3E culture were incubated for 15 min. at 37\u00C2\u00B0. The phage/bacteria solution were mixed with 8 ml molten top agarose (maintained at 42\u00C2\u00B0) and spread on the plates. After the top agarose had solidified, plates were incubated overnight at 37\u00C2\u00B0. Plaques were transferred to nitrocellulose membranes (Schleicher and Schuell) by capillary action. Duplicate filters were prepared for each plate. Filters were processed for hybridization by soaking 4 min. each in Denaturing solution (1.5 M NaCl/ 0.5 M NaOH), Neutralization solution (1.5 M NaCl/ 0.5 M Tris pH 8.0), and lx SSPE (20x stock 83 contains (in g/1): NaCl, 175.3; NaH2P04, 27.6; EDTA, 7.4). The membranes were air dried and baked for 45 min. at 80\u00C2\u00B0. After baking, the filters were incubated in prehybridization solution consisting of 5x SSPE, 0.2% SDS, 5x Denhardt's solution (50x: 5 g Ficoll type 400; 5 g polyvinylpyrrolidine; 5 g bovine serum albumin Fraction 5 in 500 ml dH20), and 0.2 mg/ml salmon sperm DNA for at least lh at 60\u00C2\u00B0 in an shaking water bath. Filters were placed in fresh prehybridization solution and incubated overnight at 60\u00C2\u00B0 overnight with the radiolabeled DNA probe. Filters were then washed in lx SSPE/ 0.1% SDS at 60\u00C2\u00B0, air dried, and exposed to film for 5 days at -80\u00C2\u00B0 in the presence of an intensifying screen. Positive plaques were extracted from the agar with the large end of a glass Pasteur pipette and placed in 1 ml A, dil. Phage from these plugs were diluted in X dil for the second round of purification. This procedure was repeated for three rounds of purification, except that after the first round, plaque lifts were not made in duplicate. Samples were considered pure when 100% of the plaques gave a hybridizing signal. Stocks of pure phage were established and stored over 2 ml of chloroform at 4\u00C2\u00B0. To excise the cDNAs from the AACT vector, a 4 ml NZYCM culture containing 0.02% maltose and 50 pig/ml kanamycin was inoculated with a single colony of RB4E and grown overnight at 37\u00C2\u00B0. 100 pi RB4E and 1 pi phage stock were combined and incubated for 30 min. at 37\u00C2\u00B0. 1 ml of 2x YT media was added, and placed in a shaking incubator set to 37\u00C2\u00B0 for 30 min. A fraction of this solution was plated on bacterial growth plates containing 100 pLg/ml Ampicillin, and single colonies were picked and analyzed. 84 Constructs generated in this study, cDNA Clones. With the exception of yk 131b 1, all cDNAs were isolated from the cDNA library A A C T -RB2 or amplified from C. elegans RNA. The CE2 clone was generated by RT-PCR. The degenerate primers CES and CEA were used to amplify sequences from C. elegans total RNA. An ~350-bp product was subcloned into T-tailed pBluescript KS generated by the method described in Collins et al. (1990). The ykl31bl cDNA was obtained as a AZapH clone from Y. Kohara at the C. elegans cDNA Project. The pBluescript plasmid containing the cDNA insert was excised following the protocol provided by Stratagene and transformed into E. Coli (XL 1-Blue strain). Plasmid DNA was prepared by alkaline lysis and sequenced using the Sequenase 2.0 kit (U. S. Biochemical Corporation). CDNA121 is a 5' RACE product. Primer EM83 was used to reverse transcribe the RNA. The nested antisense primer EM89 was used in the subsequent amplification steps. cDNA products were subcloned into the pCR2.1 T-tailing vector (Invitrogen) for analysis. CDNA82-43, cDNA92-68 and, cDNA39-52 were amplified from the AACT-RB2 library. 1 pJ AACT-RB2 (estimated titer = 107 pfu/p,l) per PCR reaction was amplified for 40 cycles of 1 min. at 95\u00C2\u00B0, 30 sec. at 49\u00C2\u00B0, and 1 min. 23 sec. at 72\u00C2\u00B0. Amplification products were subcloned into the pGEM-T Easy vector for analysis. cDNA82-43 is a 1132-bp fragment amplified with EM82 and EM43. The 859-bp fragment of cDNA92-68 was amplified with EM92 and EM68, while the 1327-bp cDNA39-52 was amplified with EM39 and EM52. Seven cDNA clones, cDNAl, cDNA6, cDNA8.1, cDNA8.2, cDNA9, cDNAlO, and cDNA15, were isolated from the AACT-RB2 cDNA library in the manner described above. 85 cDNA155#18, rh-3, and rh-4 are cDNA clones representing the predicted 5'-end of unc-2 and were obtained by RT-PCR. In the case of cDNA155#18, EM 154 was used to reverse transcribe the RNA, whereas the rh-3 and rh-4 clones were derived from samples reverse transcribed with the random hexamer oligos (BRL). Amplification proceeded as described, using the primer pair EM155-EM154., '. i Genomic Clones: The fragments used to generate the genomic clones CEC-32 and CEC-11 were identified by probing Southern blots of the cosmid T02C5 with the Hind Yil-Eco RI fragment of CE2. CEC-32 was constructed by subcloning the 2.3-kb Eco RI fragment into Eco Rl-digested pBluescript KS. CEC-11 was constructed by subcloning the 2.3-kb Bam HI fragment into pBluescript KS digested with Bam HI. CEC-1S contains a 1.9-kb Sal I fragment subcloned into the Sal I site of pBluescript KS. This fragment was identified by probing a Southern blot of T02C5 with 32P-y-dATP-labeled-EM32. Amplification of genomic DNA from unc-2(mdl 186) with the primer pair p618-EM83 yielded a 958-bp product. This PCR product was then subcloned into pGEM-T (Stratagene) to generate the Tel-3 clone. Generation of Mutant Constructs. cc]B Clones. The CMVEF construct was used as an intermediate in the construction of the CMV30-14G-612 and CMV30-14G-611 mutant clones. This construct contains the 4551-bp Bsi WVAfl II fragment from the rat brain a 1 B clone CMV30-14G (Dubel et al, 1994; Stea et al, 1995b) in pSL 1180 (Pharmacia) and was generously provided by G. Zamponi. CMVEF was further modified to remove most of the polylinker by digesting CMVEF with Nhe I and Sal I, polishing the ends with T4 DNA polymerase (BRL), and recircularizing to yield pSLEF. 86 aIB-612. The Gly to Arg mutation in the ra612 mutant was introduced into CMV30-14G using the method described in the Quikchange Site-Directed Mutagenesis Kit (Stratagene). The complementary primers EM 126 and EM 127 were designed such that the GGG codon for G1739 in CMV30-14G was altered to AGG, thereby changing the glycine residue to an arginine. Each primer was used at a concentration of 11 pmolAil. Between 5 and 50 ng of CMVEF in a volume of 10 p\l was used as the template in PCR reactions containing 5pl 1 Ox Pfu amplification buffer, 1 ixl 10 mM each dNTPs, and 1 ]\\ Pfu DNA polymerase. The final volume was adjusted to 48 p,l with dH20. The amplification buffer, dNTPs, and DNA polymerase were combined as a master mix. Template and master mix were subjected to a \"hot start\" procedure for 5 min. at 68\u00C2\u00B0, after which 38 p,l of the master mix were added to each template tube. Finally, 2 pi of a primer mix containing 1 pi each of EM 126 and EM 127 were added. The mixture was then amplified for 12 cycles consisting of 30 sec. at 95\u00C2\u00B0, 1 min. at 68\u00C2\u00B0, and 16 min. at 72\u00C2\u00B0, followed by a 5 min. incubation at 68\u00C2\u00B0. After 12 rounds of amplification, samples were ethanol precipitated. The DNA was resuspended in 8 pi dH20 and digested with Dpn I (NEB) in the appropriate reaction buffer. The samples were then extracted with phenohchloroform and precipitated with ethanol. The DNA was resuspended in 4 p,l dH20 and the full volume was used to transform Epicurian Coli XLl-Blue Supercompetent cells (Stratagene). Transformation reactions were plated on 2xYT - agar plates containing 100 u\g/ml ampicillin and a single recombinant clone (designated CMVEF-612) containing the desired mutation was identified. The 510-bp between the Sph I and Xho I recognition sites flanking the mutation were sequenced. This Sph I - Xho I fragment was then subcloned into Sph I - Xho I digested pSLEF to generate pSLEF-612. This extra cloning step ensured that no PCR-87 induced errors were introduced into the clone. The BsiWl - Afl II fragment was removed from pSLEF-612 and subcloned into CMV30-14G/ BsiWl - Afl II to yield a1B -612. a1B-611. An alternate strategy was used to introduce the glycine to arginine mutation found in the ra611 allele of unc-2 into CMV30-14G. The glycine altered in this mutant corresponds to G1578 of CMV30-13G. Two primers, one forward (EM131B) and one reverse (EM144) were designed, each of which contained the G1578 codon near the 5' end. However, the GGC codon for G1578 was changed to CGC, converting the glycine residue to an arginine, as well as introducing an Eco 47 LII site. The primer set EM131B-R18 was used to amplify a 385-bp fragment from CMV30-14G, which was subcloned into the Eco RV site of pBluescript KS. This clone, pBS.35, was sequenced between the Eco Al III site introduced by EM131B and the unique Sph I site 112-bp downstream to ensure that no sequence errors were present. The reverse primer was used with primer 081 to amplify a 1.2-kb fragment from CMV30-14G. This fragment was cloned into pGEM-T Easy to yield 611.081. Again, the sequence of the clone was confirmed by DNA sequencing. The 774-bp Pst I - Eco 47 III fragment from 611.081 was subcloned into Eco Al III - Pst I digested pBS.35, resulting in pBS611.116 which consists of a 1058-bp fragment of CMV30-14G containing the G1578R mutation. The 1086-bp Pst I fragment from pSLEF was introduced into pBS.611.116 at the Pst I site. This resulted in a clone pBS611.116P which contained two unique sites, Bsi WI and Sph I, which were used to insert the mutation into pSLEF (pSLEF-611). The 4.5-kb Bsi WI - Afl II fragment was excised from pSLEF-611 and introduced into CMV30-14G to form the full-length mutant construct a1B-611. 88 oc 1 A Clones. The rat brain ccIA clone pc3RBAl (Starr et al, 1991) was modified to facilitate introduction of mutations into the full-length construct. The polylinker at the 5' end of the clone (bases 890-971) was removed by digesting pc3RJ3Al with Hind III and Xho I and polishing the ends with T4 DNA polymerase (BRL). The construct was recircularized to yield ma1 A. A second construct, pucA(EBII), was also used as an intermediate to introduce the ra612 and ra611 mutations into ma1A. The pucl8 plasmid was digested with Eco RI and dephosphorylated using Shrimp Alkaline Phosphatase (USB). The 5.8-kb Eco RI fragment was isolated from ma, A and subcloned into the digested pucl8 to produce pucA(E5.8). The Not I and Kpn I sites located 3' of the Eco RV recognition site in pucA(E5.8) and the Kpn I site in the polylinker of pucl8 were removed by digesting with Bgl II and Nde I, polishing the ends with T4 DNA polymerase (BRL), and recircularizing to yield pucA(EBU). Once the desired mutations were introduced into pucA(EBII), they were shuttled into moc1A using the unique Nhe I and Eco RV sites. m a1A-612. The glycine mutated in the ra612 allele of unc-2 corresponds to Gl817 of pc3RBAl. Two partially overlapping primers (EM148; forward, and EM149; reverse) were designed such that the Gl 817 codon (GGC) was located near the 5' end of each primer. This codon was changed to CGT, thereby altering the glycine to an arginine. These substitutions also result in the creation of a Sna BI site at the 5' ends of each primer. The upstream primer EM148 was used with the pc3RBAl reverse primer WG to amplify a 402-bp fragment from pc3RBAl which was subcloned into pGEM-T Easy (Promega). The region of this clone (EM148WG) was sequenced between the Sna BI site introduced by EM 148 at the 5' end of the fragment and the Eco RV site 120-bp downstream to confirm that no PCR-induced errors were present. A 253-bp fragment was 89 amplified from pc3RBAl using primers 73-X and EM149 and subcloned into pGEM-T Easy. This clone, designated 73X149, was sequenced between the Sna BI site at the 3' end of the clone and the Not I site located 68-bp upstream. EM148WG was found to contain two PCR fragments in the same orientation. This enabled the construction of clone 73XWG simply by inserting the Sna BI fragment from EM148WG into Sna BI digested 73X149. The 186-bp Not I - Eco RV fragment containing G1817R from 73XWG was inserted into pucA(EBII) to yield pucA(EBII)-612. The full length clone ma 1 A - 612 was generated by inserting the Nhe I - Eco RV fragment of pucA(EBU)-612 into moc1A. Generation of Antibodies and Immunohistochemistry. Fusion Protein Construct: To construct the FP2 clone, RT-PCR was used to amplify a 177-bp fragment encoding the cytoplasmic linker connecting Domains II and III. Primers EM61 and EM62 were designed with homology to the unc-2 sequence at the 3' end. Eco RI and Sal I recognition sites were incorporated onto the 5' ends of each primer, respectively. In addition, two stop codons, TAG and TGA, were inserted between the homologous sequence and the Sal I site in EM62. Taq (BRL) and Vent (New England Biolabs) DNA polymerases were used in the amplification reaction in a ratio of 30:1 to reduce the occurrence of PCR-induced errors and to obviate the need for polishing of the PCR product before blunt-end cloning. The fragment was subcloned into pBluescript KS digested with Eco RV and colonies carrying recombinant plasmids were identified through blue-white selection. Plasmid DNA from a positive clone (pBS.FP2.1) was purified and the sequence confirmed. The insert was then excised from pBS.FP2.1 with Eco RI and Sal I and subcloned into pGEX-4T (Pharmacia). Positive clones were identified by hybridization using EM62 labeled with 32P-y-dATP as a 90 probe. Several FP2 clones were chosen for small scale induction experiments to confirm expression of the fusion protein prior to undertaking large scale purification. Fusion protein expression, purification, and analysis. To analyze expression of fusion proteins, cell lysates were prepared from cultures after induction with IPTG. 0.1 ml of overnight culture was added to 1 ml fresh medium and grown for 60 - 90 min. IPTG (Gibco BRL or Boehringer Mannheim) was added to 1 mM and the culture grown an additional 60-90 min. Cells were pelleted by centrifugation, resuspended in 50 ixl lx Laemmli sample buffer (50 mM Tris-CL (pH 6.8), 100 mM dithiothreitol, 10% glycerol, 2% SDS and 0.1% bromophenol blue), and boiled for 5 min. 4 - 5 pd of each sample was analyzed by SDS-PAGE and Coomassie staining or Western blotting. Glutathione S-transferase (GST)-conjugated fusion proteins were purified as described by Smith and Johnson (1988) with some modifications. For large scale preparations, 20 - 30 ml overnight cultures were grown at 37\u00C2\u00B0 with shaking in 2xYT containing 100 mg/ml ampicillin. 10 - 15 ml of overnight culture was added to 250 ml of fresh medium and grown for 2 - 3 hours at 37\u00C2\u00B0 with shaking. IPTG was added to a final concentration of 1 mM, and cultures were grown an additional 2-4 hours. Cells were pelleted by centrifugation at 10,000 RPM and resuspended in MT-PBS (150 mM NaCl, 16 mM Na2HP04, 4 mM NaH2P04) supplemented with PMSF and EDTA to a final volume of 9 ml. Cells were lysed by gentle sonication (6 x 30 sec. with chilling on ice between bursts). 1 ml of 10% Triton X-100 (in MT-PBS) was added and the suspension incubated for 5 min. at 4\u00C2\u00B0. Insoluble debris was pelleted by centrifugation at 10,000 RPM and the supernatant transferred to a 15 ml conical centrifuge tube containing 2 - 3 ml pre-swollen glutathione-agarose beads (Sigma Chemical Company). The tube was rotated gently for 5 min. at room temperature. Beads were washed 5x in MT-PBS + 1% Triton X-100, 91 followed by 2x in MT-PBS without detergent. Bound fusion protein was eluted 3x at room temperature with 1 volume of elution buffer (50 mM Tris-CL, 10 mM glutathione) and stored at -20\u00C2\u00B0. Generation of polyclonal antisera. To generate polyclonal antisera, New Zealand White rabbits were injected subcutaneously with purified fusion protein emulsified in Freund's complete adjuvant (approximately 0.5 mg protein/rabbit). Rabbits were boosted at approximately 4-week intervals with fusion protein emulsified in Freund's incomplete adjuvant (approximately 0.25 mg protein/rabbit) and blood samples taken 10 to 12 days post injection. Immune response was monitored by Western blotting of purified fusion proteins and immunofluorescence staining. Western blotting. Proteins were resolved by SDS-PAGE and transferred to Hybond ECL nitrocellulose membrane (Amersham) for 15 - 30 min. atl2-15Vina Trans-Blot SD Electrophoretic Transfer Cell (BioRad). Blots were blocked overnight at 4\u00C2\u00B0 in 5% milk powder-TBS-T (TBS-T: 20 mM Tris (pH 7.6), 137 mM NaCl, 0.1% Tween 20) and incubated with primary antibodies (see below) in 0.5% milk powder-TBS-T for 2 - 3 hours at room temperature. After washing in TBS-T, blots were incubated with horseradish peroxidase-labelled secondary antibodies (see below) in 0.5% milk powder-TBS-T for approximately 45 min. at room temperature. After washing in TBS-T and in TBS, blots were incubated for 1 min. in ECL detection reagents (Amersham) and exposed to film (Kodak X-OMAT). For Western blotting, rabbit polyclonal sera were diluted as follows: FP3/4 (1:5000 -1:50000) and anti-GST (1:2000 - 1:5000). The secondary antibody, horseradish peroxidase-labelled goat anti-rabbit IgG (Amersham) was diluted 1:10000. 92 Immunofluorescence staining. Larval and adult worms were stained using either a freeze-fracture procedure adapted from Albertson (1984) or the procedure described by Finney and Ruvkun (1990). These methods are also described in detail by Epstein and Shakes (1995) in \"Caenorhabditis elegans: Modern biological analysis of an organism\". For immunofluorescence staining, the rabbit polyclonal serum FP2 was diluted 1:100 - 1:500, and the mouse monoclonal antibody DM5.6 (Miller etai, 1983) was diluted 1:40 - 1:50. Secondary antibodies, FITC-labeled donkey anti-rabbit IgG F(ab')2 and TRSC-labeled donkey anti-mouse IgG F(ab')2 (Jackson ImmunoResearch Laboratories), were diluted 1:100 - 1:200. Electrophysiological recordings from HEK tsa201 cells. HEK tsa201 cells were transiently transfected with the appropriate cDN A constructs using the standard Ca2+-phosphate precipitation method. A CD8 expression plasmid was co-transfected with the VGCC subunit cDNAs. VGCC subunits ( a 1 A , o c 1 A . 6 l 2 , a 2 5 , and p l b or (32a; molar ratio 1:1:1) were co-transfected with CD8 (2 p\g) and pBluescript SK (15 p,g). A total of 20 p,g of cDNA was used per transfection. Transfected cells could be identified visually by the binding of CD8-coated Dynabeads (Dynal) (Jurman et al., 1994). Microelectrodes were constructed from borosilicate glass tubing (Sutter Instrument Co.) on a P-87 micropipette puller, and the tips were fire polished with a microforge (MF-83, Narishige). The resistance of recording electrodes was 2-5 MQ. when filled with the internal solution composed of (in mM): CsCl (105), TEAC1 (25), CaCl2 (1), and EGTA (11) and HEPES (10) pH 7.2 unless otherwise specified. In experiments in which the EGTA concentration was lowered to 1 mM, the TEAC1 concentration was increased to 30 mM to maintain osmolarity. Series resistance had typical values of 7-10 MQ and was 93 electronically compensated by at least 60%. The bath was connected to ground by a 3 M KC1 agar bridge. Currents were recorded in the whole cell patch configuration (Hamill et al, 1981). The external recording solution contained (in mM) BaCl2 or CaCl2 (5), MgCl2 (1), HEPES (10), TEAC1 (40), glucose (25), and CsCl (87.5), pH 7.4. Gigaohm seals were formed directly in the external control solution. All experiments were performed at room temperature (20 - 24\u00C2\u00B0). Recordings were performed using an Axopatch-200A or 200B amplifier (Axon Instruments, Foster City, CA), controlled and monitored with a PC 486 running pCLAMP software, version 6.03 (Axon Instruments). Data were low-pass filtered at 2 kHz using the built-in Bessel filter of the amplifier. In most cases, subtraction of capacitance an leakage currents was performed on-line using a P/4 protocol. Recordings were analyzed using Clampfit 6.03 (Axon Instruments) and figures and linear regressions were done using the software program Microcal Origin (4.1). Protocols for analysis of oc1A constructs. Cells were examined for inward currents beginning 24 hours post-transfection for cells expressing wild-type oc1A channels and 48 - 72 hours for cells expressing mutant constructs. The experiments were initiated after inward currents from cells expressing wild-type and mutant channels reached a steady-state level, which was achieved by repeatedly depolarizing the cell from a holding potential of -120 mV to 0 mV until the magnitude of the current was constant. Protocols to measure the current-voltage (I-V) relationship and steady-state inactivation (SSinact) were performed. The magnitude of the current evoked by transmembrane potential changes was measured, and expressed as an I-V relationship according to the following procedure: The membrane potential was maintained at a holding value of -120 mV and voltage test pulses of increasing magnitude were applied starting at -60 mV and increasing to +35 mV in 5 mV increments. Cells were 94 Figure 4. Protocols for electrophysiological analysis. A) Schematic of the voltage step protocol used to study the voltage dependence of steady-state inactivation. The prepulse potential at which the cell is held is indicated next to the appropriate current traces. Note that both test pulses were to 0 mV, but the amplitude of the current is maximal when the entire population of Ca 2 + channels is available (prepulse = -120 mV). When the membrane is depolarized to -50 mV during the prepulse (prepulse = -50 mV), about half of the channels are inactivated by voltage and not available to open resulting in a smaller current. B) The magnitude of current inactivation (r) was determined by dividing the current remaining at the end of the pulse (1^ ) by the maximum current obtained during the pulse (1^) to yield the percentage of current that does not inactivate during the test pulse. C) Method used to calculate the time constant (t) of inactivation for a given current trace. 95 p r e p u l s e 2 0 0 pA 2 sec held at each test potential for 150 ms. The availability of ion channels as a function of membrane potential was studied with a protocol to explore the steady-state inactivation (Figure 4A). SS jnact analysis was achieved by varying the value of a long prepulse and stepping to a constant test potential. Prepulses lasting 15 s began at -120 mV and were increased in increments of 10 mV to +70 mV. Cells were then stepped from the holding potential (-120 mV) to the test potential for 200 ms. The test potential, derived from the I-V curve, is the voltage at which peak current was obtained (i.e. 0 mV (oc1A) or +5 mV (a1A. 612) in 5 mM Ba2+). Protocols for analysis of a 1 B constructs. Cells were examined for inward currents beginning 24 hours post-transfection for cells expressing wild-type a 1 B channels and 48 - 72 hours for cells expressing mutant constructs. Protocols to measure the I-V relationship and SS inact were also performed on constructs expressing the wild-type and mutant oc1B constructs. To determine the I-V relationships of the wild-type and mutant a 1 B, the membrane potential was maintained at a holding value of -120 mV and voltage test pulses of increasing magnitude were applied starting at -30 mV and increasing to +65 mV in 5 mV increments. Cells were held at each test potential for 150 ms. In the SS inact protocols, prepulses lasting 15 s began at -120 mV and were increased in increments of 10 mV to +20 mV. Cells were then stepped from the holding potential (-120 mV) to the test potential for 200 ms. Data analysis. Only cells expressing currents between 500 and 800 pA were included in the analysis. Capacitance and leakage currents were subtracted prior to analysis using PCLAMP software to determine the magnitude, kinetics, and voltage-dependent properties of the whole-cell currents. Currents were normalized by dividing the current magnitude by the 97 maximum current recorded (/(step/ / (max)). Activation data were fitted with the Boltzmann equation: I={Gmax(Vm-Er)}/{ l+exp[(Vm-V50)/k]} SS inact data were fitted with smooth curves according to the Boltzmann equation : MM={i4\u00C2\u00ABxp[(v-v50yk l]-i where: I = the current recorded at each test depolarization (normalized 7 ( m a x ) = the maximum current recorded (normalized) Vm = the step potential (activation protocols) or the prepulse potential (SSinact protocols). V 5 0 = the potential at which I was half-maximal. G m a x = the maximum conductance E r = the reversal potential of the conducting ion k = Boltzmann factor (slope of the activation curve) The magnitude of inactivation (r) was determined by dividing the current remaining at the end of the pulse (Iped) by the maximum current obtained during the pulse (1^) to yield the percentage of current that does not inactivate during the test pulse (Figure 4B). The time constant of inactivation (x inact) (Figure 4C) was determined by calculating the best fit line according to the formula: I = I 0 + I0e\"t/T, where: I = the current I0 = the maximum current t = time Significant differences were determined using a Student's t- test with the significance value set at P < 0.01. The values given in the text and figures are mean \u00C2\u00B1 SEM. 98 Chapter 3. The unc-2 gene encodes a non-L-type VGCC aL subunit. Background. A major goal of VGCC research is to understand the contribution of specific channel sequences and structures to their functional properties. This goal was initially addressed by comparing the primary sequences of VGCCs with Na+ and K + channels. For example, the regions that form the voltage sensor and the channel pore were elucidated in this manner (see Structure and Function Studies; reviewed in Yellen, 1993). This type of analysis was expanded by the cloning of additional members of the VGCC family. Regions of sequence similarity between VGCCs with distinct physiological roles were predicted to contribute to general VGCC structure and function. In contrast, regions of sequence divergence were predicted to determine functional properties specific to different channel types. These hypotheses were tested by altering the regions in question using site-directed mutagenesis or by constructing chimeric channels. Alterations in channel behavior were then assessed by expressing the mutant channels in heterologous expression systems such as Xenopus oocytes and mammalian cell lines. Studies of VGCC structure-function relationships have also benefited greatly from the analysis of naturally occurring splice variants. Alternative splicing of a, subunit genes generates multiple isoforms of each channel class (Perez-Reyes et al, 1990; Hui et al., 99 1991; Williams etal, 1992b; Ihara et al, 1995; Kollmar et al, 1997; Soldatov etal, 1997; Soldatov et al, 1998; Stea et al, 1999; Bourinet et al, 1999). These splice variants, which often differ by only a few amino acids, display markedly different functional properties when expressed in heterologous expression systems, and these observations have provided insight into the importance of regions of the protein that had not previously been studied. Genetic studies can provide a useful complement to molecular and electrophysiological techniques in dissecting structure-function relationships in channel proteins. By examining mutants lacking a particular protein, the role of that protein in normal cellular processes can be determined. In some cases, mutations result in the expression of proteins with altered biochemical, electrophysiological, or pharmacological properties. By correlating these changes with the underlying sequence alterations, residues critical to protein function can be identified. Recently, identification and analysis of mutations in human and mouse VGCCs have provided insight into the role of these proteins in both normal physiology and disease states (Tanabe et al, 1988; Fletcher et al, 1996; Bech-Hansen et al, 1998; Kraus et al, 1998; Strom et al, 1998; Hans et al, 1999). However, prior to this study, the only known mutation affecting a VGCC was the mdg (muscular dysgenesis) mutation in mice (Beam et al, 1986; Tanabe et al, 1988; Knudson et al, 1989). A system in which mutations can be induced would be an especially useful resource; one would not have to rely on naturally-occurring mutations that are rare and may be difficult to identify. In addition, many compounds are available that produce different mutagenic events, making it possible to tailor screens to suit the need. C. elegans provides a convenient system in which mutations can be easily generated, isolated, and analyzed (reviewed in Riddle et al, 1997). When this project was initiated, a VGCC a, subunit gene had not been identified in C. elegans. However, there was some evidence that VGCCs were present in this organism. Studies on the dorsal and 100 ventral nerve cords of Ascaris, a related nematode, revealed cobalt-sensitive transients believed to be Ca 2 + currents (reviewed in Chalfie and White, 1988; Davis and Stretton, 1989[a,b]). Thus, while it was not possible (at that time) to perform electrophysiological recording from neurons in C. elegans, the results of electrophysiological studies on Ascaris suggested that VGCCs were present. More direct evidence came from studies on C. elegans itself. Willett et al. (1991) demonstrated high affinity DHP-binding sites in C. elegans homogenates, suggesting the presence of L-type VGCCs. Furthermore, the unc-36 gene has been cloned and found to encode a polypeptide with extensive similarity to the a2/8 subunit of the VGCC complex (Lobel and Horvitz, 1993). Results. Cloning and sequencing of a VGCC a, subunit in C. elegans. To identify a VGCC a, subunit-encoding gene in C. elegans, degenerate primers (CES and CEA; Table 4) corresponding to the highly conserved domain IVS5-IVS6 region of mammalian VGCCs were designed taking into account C. elegans codon usage. These primers were used to amplify homologous sequences from total C. elegans RNA by reverse transcription PCR (RT-PCR). A PCR product of approximately 350-bp was obtained and subcloned into the plasmid Bluescript. The DNA sequence was determined and the predicted amino acid sequence of the 327-bp fragment (Ce2) was compared to the analogous regions of mammalian voltage-gated Na+ and Ca 2 + channel pore forming subunits (a and a, subunits, respectively) (Figure 5). The greater sequence similarity of Ce2 to the VGCC subunits as compared to the Na+ channel subunits suggested that Ce2 encoded a VGCC a, subunit from C. elegans. 101 Figure 5. Alignment of the Ce2 sequence with the homologous region of other voltage-gated Na+ and Ca 2 + channels. The predicted amino acid sequence of the Ce2 PCR product was compared to the analogous region of the mammalian HVA VGCC subunits A through F, the C. elegans L-type channel EGL-19, and the Na+ channels RNaBl, RNaB2, and RNaB3. VGCC subunits A through E are from rat brain, while the cc1F is from the human sequence. Areas of shading indicates regions of amino acid similarity. The hatched shading indicates regions of amino acid similarity between UNC-2 and the Na+ channels but NOT the VGCCs. The hatched bars below the sequence represent the residues spanned by the CES and CEA primers. The arrow indicates the residue in the Domain IV P-loop responsible forf the ion-selectivity of Ca 2 + and Na+ channels. Overall, Ce2 is more similar to the VGCC sequences than to Na+ channels. GenBank Accession Numbers for VGCCs: rat oc1A, M64373; rat oc1B, M92905; rat 0CiC, M67515; rat oc1E, L15453; human a 1 F, AJ224874; rabbit oc]S, M23919; rat a 1 D and UNC-2, E. Mathews and T. P. Snutch, unpublished results; EGL-19, AF023602; GenBank Accession Numbers for rat brain voltage-gated Na+ channels: RNaBl, X03638; RNaB2, X03639; RNaB3, X00766. 102 IVS5 alpha 1A I T alpha 1B 1 Y A 1 1 G alpha 1E 1 Y A 1 1 G alpha 1C 1 Y A V 1 G alpha 1D 1 Y A V 1 G alpha 1F 1 Y A V 1 G EGL-19 1 Y A V 1 G rNaB1 1 Y A \u00E2\u0080\u00A2 F G rNaB2 1 Y A \u00E2\u0080\u00A2 F G rNaB3 1 Y A IF G UNC-2 1 Y A 1 V G M Q V M Q V M Q V MQM MQM M Q F Is N IG B D IE D E D S D E D E F Q I T E H N N F R T F F Q F G N I A L D D G M S I N R H N N F R T F L Q F G N I K L D E E S H I N R H N N F R S F F G F G K I A L N D T T E I N R N N N F Q T F P Q F G K V A M R D N N Q I N R N N N F Q T F P Q F G K V A L Q D G T Q I N R N N N F Q T F P Q F G K V A I D D S T S I H R N N N F H S F P A F A Y V K R E V G I D D M F N F E T F G N F A Y V K R E V G - I D D M F N F E T F G N F A Y V K K E A G I D D M F N F E T F G N F G N I W L N A A T E I N R H N N F Q S F F N CES alpha 1A F Q A L M L L FR alpha 1B L Q A L M L L FR alpha 1E F G S L M L L FR alpha 1C P Q A V L L L FR alpha 1D P Q A V L L L FR alpha 1F F P Q A V L L L FR EGL-19 F P A A I L V L FR rNaB1 G N S M I C L F Q rNaB2 G N S M I C L FQ rNaB3 J3 N S M I C L FQ UNC-2 F F N A V I L L FR s i s j C A C A C A S A T T A WH N A WH E A WQE A WQD A WQE A WQE G E A W Q D S A GjglDG ^ WD G WQD V Q G K K N S G I Q K - -P H A N A S P D T T A P S - -P E S E P - - S N P D S D Y P E S D F P U S D D Y - H K D C D P N K V N P - - G D C D P E K D H P - - G D C D P N A I H P - - G D C A R A G S A E I N -alpha 1A alpha 1B alpha 1E alpha 1C alpha 1D alpha 1F EGL-19 rNaB1 rNaB2 rNaB3 UNC-2 Q N E S T K G E P G E E P G E E G L N E S V K G S V K G S V K G F E K G IVS6 Q T CG S CEA 103 Ce2 was then used as a probe to map and further characterize the corresponding VGCC gene. Southern blot analysis of C. elegans genomic DNA digested with various restriction enzymes showed that in most cases Ce2 hybridized to a single restriction fragment (Figure 6). This suggested that the Ce2 VGCC a, subunit is encoded by a single gene in C. elegans. This hypothesis was confirmed by screening a genomic YAC grid which represents the entire C. elegans genome (Coulson etai, 1991). The Ce2 probe hybridized to three overlapping YACs (Y76F7, Y23A3, and Y5E3) from the left arm of the X chromosome (Figure 7A). The region covered by these YACs is represented by seven cosmid clones (M01A9, C44B3, F54G2, C09C10, T14G11, T02C5, and W10B11; Figure 7B). Each of the cosmids was grown up and DNA isolated. Hybridization with the radiolabeled Ce2 probe revealed that a single cosmid, T02C5, contains the VGCC sequences. To confirm that T02C5 contained the Ce2 sequences, the T02C5 DNA was digested with various restriction enzymes and probed with radiolabeled Ce2. Figure 8 shows that Southern blotting of the T02C5 cosmid revealed an identical pattern of hybridization to the C. elegans genomic blot (compare to Figure 6), confirming that T02C5 carried the VGCC sequence. This result also indicates that the T02C5 cosmid has not undergone any major rearrangements. Taken together, these results indicate that the VGCC corresponding to the Ce2 PCR product is located on the X chromosome in the vicinity of the unc-2 and dpy-3 genes. This VGCC was subsequently shown to correspond to the unc-2 gene isolated genetically by S. Brenner (Schafer and Kenyon, 1995). To obtain additional genomic sequence, several restriction fragments from the T02C5 cosmid were subcloned and sequenced: i) the clone CEC-32 contains a 2.3-kb Eco RI fragment, ii) the CEC-11 clone contains a 2.3-kb Bam HI fragment that overlapped with the 3' end of CEC-32, and iii) clone CEC-IS contains a 1.9-kb Sal I fragment. This third fragment was identified by probing a Southern blot of T02C5 fragments with an oligonucleotide generated from the sequence of a 2.3-kb Eco RI fragment (CEC-bs.e) 104 Figure 6. Genomic Southern Blot of Ce2/UNC-2. The Ce2 PCR fragment was used as a probe to localize the Ce2/UNC-2 VGCC in the C. elegans genome. Southern blot analysis of C. elegans genomic DNA suggests that Ce2 represents a single copy element in the C. elegans genome. Restriction enzymes used were: B) Acc I, C) Aga I, D) Bam HI, E) Bgl I, F) Bgl II, G) Cla I, H) Eco RI, I) Eco RV, J) Hind III, K) Kpn I, L) Pst I, M) Sal I, N) Sma I, O) Sst I, P) X&a I, Q) Xho I. Lanes A and R) X Hind III molecular weight marker. 105 o r i g i n -6.5 kb 4.3 kb 2.3 kb 2.0 kb 106 Figure 7. Genomic Localization of Ce2/UNC-2. A) The Ce2 probe hybridized to three overlapping YACs (Y5E3, Y23A3, and Y76F7) which are indicated by the arrow. B) YACs are indicated by boxes and the underlying cosmids by lines. Analysis of these cosmids revealed that T02C5 hybridized with Ce2 and contains most of the corresponding gene. The remainder of the gene is not represented on cosmids and was obtained from the C. elegans Genome Sequencing Consortium. 107 A Figure 8. Southern blot of cosmid T02C5. The Ce2 PCR fragment was used to probe a Southern blot of restriction endonuclease-digested T02C5 cosmid DNA. Restriction enzymes used were: A) Acc I, B) Bam HI, C) Cla I, D) Eco RI, E) Pst I, F) Sal I, (+) plasmid Ce2 positive control. 109 (+) A B C D E F origin 4|i ... 2.0 kb 1.0 kb 0.5 kb 110 provided by W. R. Schafer (data not shown). Together the CEC-32, CEC-11, CEC-bs.e, and CEC-1S fragments (designated CEC-1S-11) represent 7.0 kb of genomic sequence. The CEC-1S-11 sequence was translated in all three reading frames and intron-exon boundaries identified by comparison with the consensus splice acceptor and donor sequences (Emmons, 1988). The CEC-1S-11 genomic sequence contains twelve complete and two partial exons that together comprise 3,489-bp of coding sequence (Figure 9). The remaining 3,583-bp are contained in thirteen introns. CEC-IS contains sequence that encodes all of domain n, as well as part of the cytoplasmic loop linking domains I and II. The 219 amino acids encoded by the portion of the CEC-bs.e clone not covered by CEC-S1 constitute the II-IIJ loop and the first four segments of domain III. CEC-32 encodes 488 amino acids beginning at the carboxyl end of domain IIIS4, past domain IVS6, extending 36 residues into the carboxyl terminus. CEC-11 exons comprise an additional 188 residues in the carboxyl terminus. The predicted coding sequence represented by these fragments was compared with that of the cloned mammalian a, subunits (Stea et al, 1995a). Consistent with the results of the Ce2 alignments, CEC-1S-11 also bears a high degree of similarity to the analogous region of mammalian VGCCs and is most closely related to the DHP-insensitive class A, B, and E a, subunits (71%, 72%, and 70% amino acid similarity, respectively). The remainder of the cosmid T02C5 was sequenced and annotated by the C. elegans Genome Sequencing Consortium (C. elegans Sequencing Consortium, 1998). The sequence contained in this cosmid extends 5' only as far as Domain IS4. Since the adjacent cosmid F25H10 does not contain any unc-2 sequence, the amino terminus and first three membrane-spanning segments of domain I must lie in the gap between T02C5 and F25H10. Partial sequence of the unc-2 gene contained in this gap was obtained by analyzing two overlapping clones, Tcl-3 and cDNA121, generated through the analysis of unc-2(mdl186) (see Chapter 4) and 5' RACE experiments, respectively. The Tcl-3 111 construct contains 966-bp of DNA amplified from unc-2(mdl186) and subcloned into the pGEM-T vector. The 3' end of Tcl-3 overlaps with the 5' end of T02C5 and the remainder of the clone contains an additional 632-bp of new sequence. The cDNA121 clone extends 5' of Tcl-3 by an additional 30-bp (10 amino acids). The majority of Tcl-3 (488-bp) is intronic sequence, as determined through analysis of cDNA121. The exons encode the IS3 segment and the adjacent cytoplasmic loops. The complete genomic structure of the unc-2 gene. The unc-2 gene spans 25.2 kb of genomic sequence and consists of 28 exons and 27 introns (Figure 9). The exons range from 47-bp (exon 1) to 879-bp (exon 28), with an average length of 239-bp. The variation in intron size is even more pronounced. The majority of introns in unc-2 are between 42- and 60-bp; however, introns 1 and 2 are 5.4 and 4.7 kb, respectively. With the exception of intron 8, all introns conform to the GU-AG splice site consensus sequence. Intron 8 begins with a GC dinucleotide, which has also been noted, albeit rarely, at the 5' splice site in C. elegans (reviewed in Blumenthal and Steward, 1997). Exon 1 contains a putative initiator ATG methionine. Exon 28, determined through cDNA sequence analysis, contains an in-frame termination codon (TAG) and a potential polyadenylation sequence (AATAAA) 688-bp downstream. Several cDNAs were isolated and sequenced to confirm the predicted intron-exon boundaries (Figure 9). One of these cDNAs, ykl31bl, was provided by Y. Kohara of the C. elegans cDNA Project. This cDNA is -3.4 kb in length and extends from exon 17 to the 3' end of the gene. This cDNA includes a termination codon followed by a 708-bp 3' untranslated region and a poly (A) tail. A polyadenylation consensus sequence is located 15-bp upstream of the poly (A) tail. Four overlapping cDNA clones representing the remaining ~3 kb of unc-2 coding sequence were isolated in a screen of a AACT library provided by R. J. Barstead and by RT-PCR. The 5'-most of these clones, cDNA155#18, 112 Figure 9. Structure of the unc-2 gene and protein products. The unc-2 gene consists of 28 exons and spans over 25 kb. Exons and introns are represented as boxes and lines, respectively. Shown above the gene are the four genomic clones generated in this study. The unc-2 gene encodes a protein of 1992 amino acids that is most closely related to the N- and P/Q-type VGCC a, subunits expressed in the mammalian nervous system. Like other VGCC a, subunits, UNC-2 has four homologous domains (I-IV), each containing six 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 UNC-2 and were used to verify intron-exon boundaries. 113 114 was amplified from adult C. elegans RNA using EM 155, a primer designed against the putative 5' end of the gene, and EM83, which is homologous to a sequence previously shown to be present in the unc-2 coding region. This clone is 771-bp in length and encodes the amino terminal region, the first five transmembrane segments of domain I, and a portion of the IS5-IS6 pore-forming loop. A second cDNA clone of 1151 -bp was amplified from the AACT-RB2 cDNA library. This clone, cDNA82-43, overlaps with the 3' end of cDNA155#18 and extends 911-bp further downstream (up to IIS3). Finally, cDNAl was isolated from AACT-RB2 in a library screen. This clone contains almost 3.3 kb of coding sequence and encodes the carboxyl terminal half of IIS2, past IVS6, extending 233 amino acids into the carboxyl tail. The results obtained from these clones were confirmed through the analysis of eight additional cDNAs derived either from the library screen or through PCR. The primary structure of the UNC-2 VGCC a, subunit. The longest open reading frame of unc-2 encodes a 1992-amino acid polypeptide with a predicted molecular weight of 227 kDa. The predicted sequence is shown in figure 10. The primary sequence of UNC-2 is similar to that of other cloned VGCCs. Like other VGCC a, subunits, UNC-2 has four homologous domains (I-IV), each containing six hydrophobic membrane-spanning segments (S1-S6). These predicted transmembrane domains are very similar to the analogous regions of other VGCCs, especially in the voltage-sensing S4 segment and the pore forming S5-S6 loop (P-loop). In addition, a number of other features are well conserved, including the binding motifs for the p subunit (Pragnell et al, 1994) and G p y (De Waard et al, 1997; Zamponi et al, 1997) in the I-II linker, the EF-hand motif in the carboxyl-terminal region (Babitch, 1990), and four conserved glutamate residues, one in each P-loop, that determine the ion-selectivity of the channel (Heinemann et al, 1992, Yang et al, 1993). There are 49 consensus sites for 115 Figure 10. Complete amino acid sequence of the UNC-2 VGCC a , subunit. Transmembrane segments are indicated with lines above the sequence and conserved motifs with lines below. Potential N-linked glycosylation sites are indicated with (+) and putative phosphorylation sites are indicated with the following symbols: # = calmodulin-dependent protein kinase II and cAMP-dependent protein kinase; t = cAMP-dependent protein kinase; f = cAMP-dependent protein kinase and protein kinase C; $ = protein kinase C; \u00E2\u0080\u00A2 = calmodulin-dependent protein kinase II, cAMP-dependent protein kinase, and cGMP-dependent protein kinase. Amino acids in bold (E) indicate the coordinating glutamate residues responsible for Ca 2 + selectivity. 116 M I K E A V Q M A V W P A L P R L A A E E A R R E Q K A E S G T F V R K T T L S S N A P V K E K G P S S L F I F A E D N 6 0 I S l I S 2 I I R R N A K A I I E W G P F E Y F I L L T I I G N C W L S M E Q H L P K N D K K A L S E W L E R T E P Y F M G I F C 120 ( I S 2 ) I S 3 . _ LECVLKVIAFGFALHKGSYLRSGWNIMDFIVWSGWTMLPFSPATQTANQPVDSVDLRT 180 IS4 IS5 LRAVRVLRPLKLVSGIPSLQWLKSILCAMAPLLQIGLLVLFAIIIFAIIGLEFYSGAFH 2 40 + + SACYNERGEIENVSERPMPCTNKTSPMGVYNCDVKGTTCLQKWIGPNYGITSFDNIGFAM 3 00 + I S 6 ITVFQCITMEGWTTVMYYTNDSLGSTYNWAYFIPLIVLGSFFMLNLVLGVLSGEFAKERE 360 t RVENRREFLKLRROOOIERELNGYLEWILTAEEVILKEDRTTEEEKAAIMEARRRAANKK 42 0 (i subunit binding region LKQASKQQSTETEEDFEEDEDEMEEEYVDEGGTVEDEFAERKKRGCCHSVGKFIKQLRIQ 480 IIS1 IIS2 IRIMVKTQIFYWSVITLVFLNTCCVASEHYGQPQWFTDFLKYAEFVFLGIFWEMLLKLF 540 IIS3 IIS4 $ AMGSRTYFASKFNRFDCWIVGSAAEVIWAEVYGGSFGISVMRALRLLRIFKLTSYWVSL 600 $ IIS5 RNLVRSLMNSMRSIISLLFLLFLFILIFALLGMQLFGGRFNFPTMHPYTHFDTFPVALIT 660 IIS6 VFQILTGEDWNEVMYLAIESQGGIYSGGWPYSIYFIVLVLFGNYTLLNVFLAIAVDNLAN 72 0 AQELTAAEEADEKANEIEEESEELDEQYQEGDHCTIDMEGKTAGDMCAVARAMDDLDEEC 7 80 I I I S 1 EEEESPFGGPKPMVPYSSMFFLSPTNPFRVLIHSIVCTKYFEMMVMTVICLSSVSLAAED 840 IIIS2 I I I S 3 PVDEENPRNKVLQYMDYCFTGVFACEMLLKLIDQGILLHPGSYCRDFWNILDGIWTCAL 900 I I I S 4 $ FAFGFAGTEGSAGKNLNTIKSLRVLRVLRPLKTIKRIPKLKAVFDCWNSLKNVFNILIV 960 I I I S 5 YFLFQFIFAVIAVQLFNGKFFFCTDKNRKFANTCHGQFFVYDNQNDPPRVEQREWRLRPF 1020 I I I S 6 NYDNTINAMLTLFWTTGEGWPGIRQNSMDTTFEDQGPSPFFRVEVALFYVMFFIVFPFF 1080 117 ( I I I S 6 ) # $ F V N I F V A L I I I T F Q E Q G E A E L S E G D L D K N Q K Q C I D F A L N A R P R S L F M P E D K N S T K Y R I W R 1 1 4 0 # I V S 1 T V S 2 L V T S P P F E Y F I M T M I C C N T L I L M M K Y Y N N P L F Y E E I L R L F N T A L T A V F T V E S I L K I L A F G 12 00 I V S 3 I V S 4 $ V R N Y F R D G W N R F D F V T W G S I T D A L V T E F G G H F V S L G F L R L F R A A R L I R L L Q Q G Y T I R I L 12 60 $ I V S 5 LWTFVQSFKALPYVCLLIGMLFFIYAIVGMQVFGNIWLNAATEINRHNNFQSFFNAVILL 132 0 + T V S 6 FRCATGEGWQDIMMAAVQGKDCARAGSAEINFEKGQTCGSNVSYAYFTSFVFLSSFLMLN 13 80 # $ LFVAVIMDNFDYLTRDSSILGPHHLDEFIRVWADYDPAATGRIHYSEMYEMLRIMAPPVG 1 4 4 0 EF hand motif t $ FGKKCPYRLAYKHLIRMNMPVAEDGTVHFTTTLFALIRESLSIKMRPVEEMDEADEELRL 1 5 0 0 f t TLKKIWPLKAKKN1WDLVVPPNHELCFQKLTVGKIYAGLLILENYRARKSGTEVGGQGLF 15 60 G G G L R S L V A A A K A A E S Q H S S H T P Q P P E E T T P I I P Q H A Q Q F S A A P T M S A Q G S L Q Q M Q G T S S 1 6 2 0 # $ $ . . G G G Q R P Y S L F N S F V D T I K S G K Q D G D V T D V Q Y Q S V D Q Q H E K M N S T G R R L S D M F S K I R R G T S 1 6 8 0 #$ # # $ $ A D H N P H Q T E H L L A Q D N R S P S S P R Y R S M A R A S P P S P A E R Y G H P P R Y R T E S P P S S R S E Y Q M S 1 7 4 0 # t I R D P I I R R N R Y N T M E H S R S S H D P Q Y H Q D Q Q Q Q Q Q P H H Q Q H S Q H L Q H S H H K T Y Q N H N Q Y S R 1 8 0 0 $ S P I Y S D D S S V A E S Y R R E R E F R R Y Q D S T P Q D V S E D D D P M P T A V R A R R L P L I S T M P T H Y E S A 1 8 6 0 $ t Y Q P S S Y N Q H L N D S Y G L G T G Y Q R D Y H T S H S H S H H P T S Q Q Q Q H Q P M Y S T S P L I S P R S S H S Y Y 1 9 2 0 $ t $ t $ # #$ T P R S S Q Y Y E I P S P S P D I Y P S Y R G S A S P R R Y P T S T V W A P D R E G S S A R V I Q A Q P G S I P L S D 1 9 8 0 S E T E D D P R W A I V 1992 1 1 8 phosphorylation by calmodulin-dependent protein kinase II, PKA, PKC, and cGMP-dependent protein kinase (Kreegipuu et al, 1999). Thirty-nine of these sites are located in predicted cytoplasmic regions of the protein; thirty of these sites are located in the carboxyl tail, and a putative PKA consensus site is present in the I-II linker near the (3 subunit binding motif. Of the nine N-glycosylation consensus sites (N-X-S/T) in UNC-2, four are located in predicted extracellular regions. UNC-2 is most similar to the other VGCCs in the four transmembrane domains while differing considerably in the intracellular II-IIJ loop and carboxyl terminal regions. UNC-2 is more closely related to the DHP-insensitive a, subunit classes (B, 73%; A, 67%; E, 66% similar) than the L-type channels, which are approximately 55% similar at the amino acid level (Table 5). Furthermore, a defining feature of L-type channels is their sensitivity to DHPs (Moreno, 1999), and the channel regions involved in DHP binding (Tang et al, 1993; Grabner et al, 1996; Schuster et al, 1996) are not present in the UNC-2 protein. These results are summarized in Figures 11 through 13. There are, however, a number of differences both between UNC-2 and the DHP-insensitive channels, and between UNC-2 and vertebrate VGCCs in general. Unlike the class A, B, and E a, subunits, which have large intracellular loops of 400 to 500 amino acids joining domains II and III, the analogous region in UNC-2 is more similar in size (104 amino acids) to the II-ILT linkers found in the DHP-sensitive L-type channels. In addition, UNC-2 has a unique pattern of charge placement in the S4 domains. In vertebrate channels, the S4 segments in domains I, II, and IV contain five positively-charged residues, while in domain III, the S4 segment contains either six (DHP-sensitive channels) or seven (DHP-insensitive channels) positive charges. In the UNC-2 protein, the S4 segments in domains I and III are almost identical to the homologous regions of the DHP-insensitive channels; the few amino acid differences are conserved changes and UNC-2 contains the extra positive charge in IIIS4. In contrast, domains IIS4 and IVS4 of UNC-2 119 Table 5. Amino Acid % identity/similarity between V G C C at subunits Channel Type a, subunit C. elegans UNC-2 C. elegans EGL-19 C. elegans C54D2.5 HVA DHP-insensitive <*1A 57/67 44/57 25/38 oc1B 58/73 44/61 24/38 57/66 44/60 25/39 unc-2 100 40/54 21/35 HVA DHP-sensitive 41/54 51/63 23/37 \u00C2\u00AB 1 D 41/54 51/63 23/37 OC1F 41/55 52/63 22/36 egl-19 a, 41/55 100 23/37 <*1S 40/55 52/64 22/36 LVA T-type channels oc1G 27/46 29/48 52/68 CC1H 27/47 29/48 50/65 28/45 30/47 46/57 C54D2.5 22/36 23/36 100 120 Figure 11. Similarity tree of VGCC a1 subunits. The predicted amino acid sequences of representatives of each class of VGCC a, subunit and Na+ channel a subunits were compared pairwise and the percent similarities were plotted. GenBank Accession Numbers for VGCCs: rat a 1 A, M64373; rat a 1 B , M92905; rat oclc, M67515; rat oc1E, L15453; human a 1 F , AJ224874; rabbit ocls, M23919; rat a I D and UNC-2, E. Mathews and T. P. Snutch, unpublished results; EGL-19, AF023602; GenBank Accession Numbers for rat brain voltage-gated Na+ channels: RNaBl, X03638; RNaB2, X03639; RNaB3, X00766. The C54D2.5 sequence was annotated by the C. elegans Genome Sequencing Consortium. 121 a I S a, F 0C1D 0C1C EGL-191 r\u00E2\u0080\u0094 L - L y P e \u00E2\u0080\u00A2UNC-2-' O^lA \u00E2\u0080\u0094 \u00E2\u0080\u00A2 oc1B _ \u00E2\u0080\u00A2 a 1 E _ N-, P/Q-type P/Q-type N-type R(?)-type C54D2.5 oc1H (X 1 G T-type + + + 20 40 60 80 Percent similarity rNaB3 rNaB2 rNaBl 1 100 122 Figure 12. Alignments of UNC-2 amino acid sequence with mammalian DHP-insensitive V G C C s . The predicted amino acid sequence of UNC-2 was aligned with the amino acid sequences of the DHP-insensitive (non-L-type) VGCC a, subunits from rat brain using the Mac Vector Alignment Program. Alignments were imported into the SeqVu 1.1 Program. Shading indicates regions of sequence similarity. GenBank Accession Numbers: rat oc1A, M64373; rat a1 B, M92905; rat a,E, L15453. 123 alphalA MA R F G D E MP GR Y G A G G G G S GP A A G VV V G A A G G R G A G G S RQ 40 alphalB MVR F G D E L G G R Y G G T G G G E R \u00E2\u0080\u00A2 A R G \u00E2\u0080\u00A2 - G G A G G A G G P G Q 34 alpha 1E 0 UNC-2 M I K E A V QMA VWP A L P R L A A E E A R R 24 alpha 1A G G Q P - G A Q R M Y K Q S M A Q R A R T M A L Y N P I P V R Q N C L T V N R S 79 a lpha lB GG L-P P G Q R V I Y K Q S I AQR A R TMA L YNP I P V K Q N C F T V N R S 74 alphalE M A L Y N P I P V R Q N C F T V N R S 19 UNC-2 E Q K A E S G T F V R K T T L S S N A P V K E K G P S S 52 alpha 1A L F L F S E D N V V R K Y A K K I T E W P P F E Y M I L A T I I A N C I V L A L 119 alpha 1B L F V F S E D N V V R K Y A K R I T E W P P F E Y M I L A T I I A N C I V L A L 114 alp ha 1E L F I F G E D N I V R K Y A K K L I D W P P F E Y M I L A T I I A N C I V L A L 59 U N C - 2 L F I F A E D N I I R R N A K A I I E W G P F E Y F I L L T I I G N C V V L S M 92 IS 2 alpha 1A E Q H L P D D D K T P M S E R L D D T E P Y F I G I F C F E A G I K I V A L G F 159 alpha 1B E Q H L P D G D K T P M S E R L D D T E P Y F I G I F C F E A G I K I I A L G F 154 alp ha 1E E Q H L P E D D K T P M S R R L E K T E P Y F I G I F C F E A G I K l V A L G F 99 U N C - 2 E Q H L P K N D K K A L S E W L E R T E P Y F M G I F C L E C V L K V I A F G F 132 IS3 alpha 1A A F H K G S Y L R N G W N V M D F V V V L T G I L A T V G T E F 191 alpha 1B V F H K G S Y L R N G W N V M D F V V V L T E I L A T A G T D F 186 alp ha 1E I F H K G S Y L R N G W N V M D F I V V L S G I L A T A G T H F N T H 134 U N C - 2 A L H K G S Y L R S G W N I M D F I V V V S G V V T M L P F S P A T Q T A N Q P 172 IS 4 alpha 1A - - - - D L R T L R A V R V L R P L K L V S G I P S L Q V V L K S I M K A M I P 227 alpha 1B - - - - D L R T L R A V R V L R P L K L V S G I P S L Q V V L K S I M K A M V P 222 alp ha 1E - - - V D L R T L R A V R V L R P L K L V S G I P S L Q I V L K S I M K A M V P 171 U N C - 2 V D S V D L R T L R A V R V L R P L K L V S G I P S L Q V V L K S I L C A M A P 212 IS5 alp ha 1A L L Q I G L L L F F A I L I F A I I G L E F Y M G K F H T T C F E E G T D D I Q 267 alpha 1B L L Q I G L L L F F A I L M F A I I G L E F Y M G K F H K A C F P N S T D A E P 262 alpha 1E L L Q I G L L L F F A I L M F A I I G L E F Y S G K L H R A C F M N N S G I L E 211 U N C - 2 L L Q I G L L V L F A I I I F A I I G L E F Y S G A F H S A C Y N E R G E I E N 252 alpha 1A G - E S P A P C G T E E P A R T C P N G T K C Q P Y W E G P N N G I T Q 302 alpha 1B - - V G D F P C G K E A P A R - - - - L C D S D T E C R E Y W P G P N F G I T N 296 alpha 1E G F D P P H P C G V Q G C P A G Y E C K D W I G - P N D G I T Q 242 U N C - 2 V S E R P M P C T N K T S P M G V Y N C D V K G T T C L Q K W ' I G P N Y G I T S 292 alp ha 1A F D N I L F A V L T V F Q C I T M E G W T D L L Y N S N D A S G N T W N W L Y F 342 alpha 1B F D N I L F A I L T V F Q C I T M E G W T D I L Y N T N D A A G N T W N W L Y F 336 alp ha 1E F D N I L F A V L T V F Q C I T M E G W T T V L Y N T N D A L G A T W N W L Y F 282 U N C - 2 F D N I G F A M I T V F Q C I T M E G W T T V M Y Y T N D S L G S T Y N W A Y F 332 rs6 alpha 1A I P L I I I G S F F M L N L V L G V L S G E F A K E R E R V E N R R A F L K L R 382 alpha 1B I P L I I I G S F F M L N L V L G V L S G E F A K E R E R V E N R R A F L K L R 376 alp ha 1E I P L I I I G S F F V L N L V L G V L S G E F A K E R E R V E N R R A F M K L R 322 U N C - 2 I P L I V L G S F F M L N L V L G V L S G E F A K E R E R V E N R R E F L K L R 372 124 alpha 1A alpha 1B alp ha 1E UNC-2 alpha 1A alpha 1B alp ha 1E UNC-2 R QQQ R Q Q Q R QQQ R Q Q Q E R E E R E E R E E R E N G YMEW N G Y L E W NG YRAW N G Y L E W S K A E E FK A E E D K A E E L T A E E V I L K E D R T T E E E K A A I V I LA V M L A V M L A E D E T D V E Q R H P E E D K N A E E K S P N K N S G TS L R R A T L K K S K T D L L N P E E A E D Q L A D IAS L K R A A T K K S R N D L I H A E E G E D R F V D L C A L R R A T I K R S R T E A M T R D S S D E H C V D ISS R R R A A N K K L K Q A S K Q Q S T E T E E D F E E D E D E M E E E Y V D DGA 422 D A V 416 L E V 360 ME A 412 V G S 453 A G S 447 V G T 391 E G G 452 alpha 1A alpha 1B alp ha 1E UNC-2 alpha 1A alpha 1B alp ha 1E UNC-2 alpha 1A alpha 1B alp ha 1E UNC-2 alpha 1A alp ha 1B alp ha 1E UNC-2 P FA R A S I P FA R A S L P L A R A S I T V E DE F A E R K K R G C C H S V G K F I n s j K S A K L E N S T F F H K K E R R M R K S G K T E S S S Y F R R K E K M F R K S T K V D G A S Y F R H K E R L L R K Q L R Y I R R MV L I R R MV S R H MV Q R I MV T V L S L V A V V L C V V A I V L S V V A S V I T L V F S E M F I K M T E MS L K M L E MS L K M V E M L L K L L N T LWL L N T L C V L N T A C V L N T C C V YG L G T R YG L G P R YGMGP R F A MGS R I V H YNQP EWL S D F L Y M V H Y N Q P Q R L T T A L Y I V H H N Q P Q W L T H L L Y S E H Y G Q P Q W F T D F L K i m _ F H S S FNC F D C G V I I Y A E F F A E F Y A E F YA E F K TQA F YW K A QS F YW K S QV F YW K TQ I F YW IIS 2 I F L G L F M V F L G L F L L F L G L F L V F L G I F V F R S S FHS S FNC FNC FD F G V IV FD F G V TV F A S K FNR F D C V V IV GS I F E V I WAV I GS I F E V VWA A I GS I F E VVWA I F GS A A E V IWA E V IIS4 K P G T S FG K P G T S FG R P G T s F G Y G G - s FG I S V L R A L R L L R I F K V T K YWA I S V L R A L R L L R I F K V T K Y W N I S V L R A L R L L R I FK I T K YWA I S V M R A L R L L R I F K L T S Y W V USJ 1_ alpha 1A D T F P A A I MTV FQ L TG E DWN E V MYD E I K S QG G V QGG - MV F alpha 1B D T F P A A I L T V FQ L TG E DWNA V MYH G I E S Q G G V S K G - M F S alp ha 1E D T F P A A I MTV FQ L T G E D W N E V M Y N G I R S Q G G V S S G - MWS UNC-2 D T F P V A L I T V FQ L T G E D W N E V M Y L A I E S Q G G I YS GGWP Y alpha 1A S I Y F I V L T L F G N Y T L L N V F LA I A V D N L A N A QE L T K D E Q E E alpha 1B S F Y F I V L T L F G N Y T L L N V F L A I A V D N L A N A QE L T K D E E E M alp ha 1E A I Y F I V L T L F G N Y T L L N V F L A I A V D N L A N A Q E L T K D E Q E E UNC-2 S I Y F I V LV L FGN YT L L N V F LA I A V D N L A N A QE L T A A E E A D 493 487 431 492 533 527 471 532 573 567 511 572 L R N L V V S L L N S M 613 L R N L V V S L L N S M 607 L R N L V V S L MS S M 551 L R N L V R S L MN S M 611 alpha 1A K S I I s L L F L L F L F VV FA L L G M Q L F G G Q F N F D E G TP P TN F 653 alpha 1B K S I I s L L F L L F L F VV FA L L G M Q L F G G Q F N FQD E TP T TN F 647 alp ha 1E K S I I s L L F L L F L F VV FA L L G M Q L F G G R F N F N D G TP S A N F 591 UNC-2 R S I I s L L F L L F L F L I FA L L G M Q L F G G R F N F P T MH P Y T H F 651 692 686 630 691 732 726 670 731 1 2 5 a l p h a l A E E A A N Q K L A L Q K A K E V A E V S P L S A A N M S I A V K E Q Q K N Q K P 772 alpha 1B E E A A N Q K L A L Q K A K E V A E V S P M S A A N I S I A A R Q Q - - N S A K 764 alpha 1E E E A F N Q K H A L Q K A K E V S P M S A P N M P S I E R D R R R R H 705 E K A N E I E E 739 U N C - 2 a l p h a l A A K S V W E Q R T S E M R K Q N L L A S R E A L Y G - - D A A E R W P T T Y A R 810 a l p h a l B A R S V W E Q R A S Q L R L Q N L R A S C E A L Y S E M D P E E R L R Y A S T R 804 a l p h a l E H M S M W E P R S S H L R E R R R R H H M S V W E Q R T S Q L R R H M Q M S S Q 745 U N C - 2 E S E E L D E Q Y Q 749 alpha 1A alpha 1B alp ha 1E U N C - 2 alpha 1A alpha 1B alp ha 1E U N C - 2 P L R P D V K T H L D R P L V V D P Q E N R N N N T N K S R A P E A L R H V R P D M K T H M D R P L V V E P G R D G L R G P A G N K S K P E G T E A T E E A L N K E E A P P M N P L N P L N P L S P L N P L N A H P S L Y R R P R P G A D P P R R H H R H R D R D K T S A S T P A G G E -G L A L G L G L E K C E E E R I S R G G S L K G D I G G L T S V L D N - -846 844 I E 785 749 Q Q 886 Q D 872 Q R 822 749 a lpha 1A alpha 1B alp ha 1E U N C - 2 R T D C P K A E S T E T G A R E E R A R S P L S L G K R E P P W L P R S C H G N P R R P R R C D P E G D H T H R P V A E G E P 924 908 857 alpha 1A R R H R A R R R P - - G D E P D D R P E R R P R P R D A T R P A R A A D G E G D alpha 1B R C E R S R R H H R R G S - P E E A T E R E P R R H R A H R H A Q D S S K E G K alp ha 1E T F E D R A R H R - - - Q - . . . . S Q R R S R H R R V R T E G K E S A S A S R U N C - 2 H C T I D M E G K T A G D M C A alpha 1A D G E R K R R H R H G P P A H D D R E R R H R R R K E S Q G S G V P M S G P N -alpha 1B H S T A P V L V P K G E R R A R H R - - - - G - - - P R T G P R E alp ha 1E S R S A S Q E R S L D E G V S I D G E K E H E - - - - - - P - - - - Q S S H R -U N C - 2 V A R i M D D L D - \u00E2\u0080\u00A2 E E C E -962 947 889 768 1001 973 918 781 a lpha 1A - L S T T R P I Q Q D L G R Q D L P L A E D L D N M K N N K L A T G E P A S P H 1040 alpha 1B T E N S E E P T R R H R A K H K V P P T L E P P E R E V A E K E S N V V E G D K 1013 a l p h a l E - - S K E P T I H E E E R T Q D L R R T N S L M V P R G S \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 G L V G A L D E A 954 U N C - 2 781 alpha 1A alpha 1B alp ha 1E U N C - 2 D S L G H S G E T R N H Q P E T P L V Q P S P A K I G N S T N P G P A L A T N P Q N A A S R R T P N S L H M L P E E E S P S T C L Q K V D E L L A D V Q L D V 1079 1051 994 786 126 alp ha 1A alpha 1B alpha 1E UNC-2 N P G N P S N P G P P K T P E N S Q P E D A D N Q R N -G R G I S Q S E I V T N P S S T Q P N S A K T A R K P E H M 1119 \u00E2\u0080\u00A2 V T R M G - S Q P S D P S T T V 1076 - - P D L S C M T T N M D K A T T E S T S V 1022 786 alpha 1A alpha 1B alpha 1E UNC-2 A V E I P P H V P V T L T V A I P D A C P T G P V D P P L N H T V V Q V N K N P G E A T V V P L V D S T V V N I - - F G G A N P D P L P K K E E E K K E E E S - - - A N T D - L E G Q A E G K K E A E S N K T D G E A S P L K E A E T K E E E E -A -E E 1158 1111 1062 789 alpha 1A A D alpha 1B D D alpha 1E UNC-2 P G E D G P K P V L R R G P R P I V E K K K Q K K E K R E T G K A P K P MP V MV MV alpha 1A alpha 1B alp ha 1E UNC-2 alpha 1A alpha 1B alpha 1E UNC-2 N L R Y F E M C I L M V T M R Y F E M V I L V V N L R Y F E M C I L L V C T K Y F E MMV M T V TTTS2 JJJS_L I I Y V Y I YV Y C T G V T G V T G V F F F F T G V F A F T F E F T F E F T F E C E MV MV MV M L A MS S I A L S S I I A A S S I K M K M K M K L D L G D L G D Q G D Q G Y S Y S H S Y S L S T T N L S P T N F S T T N L S P T N L R R L C H Y L R R F C H Y I R K A C H Y F R V L I HS L V V V L A A E D P V Q P N A P R N NV L R Y F D L A A E D P V R T D S F R N NA L K Y M D L A A E D P V L T N S E R N K V L R Y F D L A A E D P V D E E N P R N K V L Q Y M D L V L H Q G A Y FR D L W N I L D F I V V L L L H P G A Y F R D L W N I L D F I V V L I L Q D G S Y F R D LWN I L D F V V V I L L H P G S Y C R D FWN I L D G I V V 1192 1145 1102 816 1232 1185 1142 856 1272 1225 1182 896 ansa IIIS4 alpha 1A S G A L V A F A F T - - - G N S K G K D I N T I K S L R V L R V L R P L K T IK alpha 1B S G A L V A F A F S S F MG G S K G K D I N T I K S L R V L R V L R P L K T IK alpha 1E V G A L V A F A L A N A L G T N K G R D I K T I K S L R V L RV L R P L K T IK UNC-2 T C A L F A F G F A G - T E G S A G K N L N T I K S L R V L R V L R P L K T IK TTTS5 alpha 1A R L P K L K A V F D C V V N S L K N V F N I L I V Y M L F M F I F A V V A V Q L alpha 1B R L P K L K A V F D C V V N S L K N V L N I L I V Y M L F M F I F A V I A V Q L alp ha 1E R L P K L K A V F D C V V T S L K N V F N I L I V Y K L F M F I F A V I A V Q L UNC-2 R I P K L K A V F D C V V N S L K N V F N I L I V Y F L F Q F I F A V I A V Q L 1309 1265 1222 935 1349 1305 1262 975 alpha 1A alpha 1B alp ha 1E UNC-2 F K G K F K G K F K G K F N G K F F H C T D E S K E F E R D C R G K Y L L Y E K - - N E V K A R D R EW F F Y C T D E S K E L E R D C R G Q Y L D Y E K - - E E V E A Q P R QW F F Y C T D S S K D T E K E C I G N Y V D H E K - - N K M E V K GR EW F F F C T D K N R K F A N T C H G Q F F V Y D N Q N D P P RV E Q R E W 1387 1343 1300 1015 127 alpha 1A alpha 1B alp ha 1E UNC-2 alpha 1A alpha 1B alp ha 1E UNC-2 KK YD FH YDNV LWA K K Y D F H Y D N V LWA K R H E F H Y D N I IWA R L R P F N Y D N T INA L L T L F T V S T G E G W P Q V L K H S V D A T F E N 1427 L L T L FTVS TGEGWPMV L K H S V D A T Y E F T V S T G E G W P Q V L Q H S V D V T E E L L M L FVV TTGE GWP G IUS$ I RQNS MD TT FE N 142 E 1383 D 1340 D 1055 QGP S P G YRME MS I QGPS PG FRME LS I R G P S R S N R M E M S I Q G P S P F F R V E V A L F Y V V Y F V V F P F F F V N I F V A L I I I F Y V V Y F V V F P F F F V N I FVA L I I I F Y V V Y F V V F P F F F V N I F V A L I I I F Y V M F F I V F P F F F V N I F V A L I I I T FQE T FQE T FQE T FQE 1467 1423 1380 1095 alpha 1 A alpha 1B alp ha 1E UNC-2 alpha 1 A alpha 1B alp ha 1E UNC-2 alpha 1 A alpha 1B alp ha 1E UNC-2 QGDK MME E QGDKVMS E QGDK MME E QGEA E LS E YRMWQ FV V YK TWT FV V YRVWH FV V YR IWR L V T A L RV FN IV M L K C L N IV A L K Y L N I A I L R L F N T A YS L E K N E R A C I D FA I CS L E K N E R A C IDFA I CS L E K N E R A C IDFA I GD LDKNQKQC I D FA L IVS1 S A K P L T R H M P Q N K Q S FQ 1507 S A K P L T R Y M P Q N K Q S FQ 1463 S A K P L T R Y M P Q N R H T F Q 1420 N A R P RS L F MP E D K N S T K 1135 S P P F E Y T I M A M I A L N T I V L M M K S P P F E Y F I M A M I A L N T V V L M M K S P S F E Y T I MAM I A L N T V V L M M K S P P F E Y F I M T M I C C N T L I L M M K IV\u00C2\u00A32 F T S L F S L E C V L K V M A F G I L N Y F F T S M F S L E C I L K I I A F G V L N Y F F T M V F S L E C V L K V I A F G F L N Y F L T A V F T V E S I LK I LA F G V R N Y F F Y G A S V A Y E F Y D A P Y E Y E Y YS A PWT YE Y YN N P L F YE HVS2^ R DAWN I F D FV R DAWNV FD FV R D TWN I F D F I R D GWN R F D FV N 1547 L 1503 L 1460 E 1175 1587 1543 1500 1215 alpha 1 A alpha 1B alp ha 1E UNC-2 IVS3 T V L G S TV LGS T V I GS TVV G S I TD I TD I TE I L V T E F G N N - - - F I L V T E I A N N - - - F I N L S F L R I N L S I I L T D S K L V N T S G F N M S L R L K TDA L V T E FGGH - F V S L G F L R IVS4 FR AA R F R A A R F R A A R FRA A R IVS5 I K L L R I K L C R I K L L R I R L L Q * alpha 1A QG Y T I R I alpha 1B QG Y T I R I alp ha 1E QG Y T I R I UNC-2 QG Y T I R I alpha 1A FGN I G I D alpha 1B F GN I A L D alp ha 1E FGN I K L D UNC-2 FGN I WL N alpha 1A TGE AWH N alpha 1B TGE AWH E alp ha 1E TGE AWQ E UNC-2 TGE GWQ D LWT FV QS LWT FV QS LWT FV QS LWT FV QS K A L K A L K A L K A L DGTS E E S H A A T E M L S C L S G K P C D MLSC LGNRACD MLSC L G E K G C E K N S G I P H A N A P D T TA QK S P S GQN E S E 1624 1580 1540 1252 Y V C L L I A M L F F I Y A I I G MQ V 1664 YV C L L I A M L F F I Y A I I G MQ V 1620 YV C L L I A M L F F I Y A I I G MQ V 1580 YV C L L I G M L F F I YA I V G MQ V 1292 T E H N N FR T F FQA L M L L FRS A 1704 N R H N N F R T F L Q A L M L L F RS A 1652 N R H N N FRS F F G S L M L L F R S A 1612 N R H N N FQS'F F N A V I L L FRCA 1324 I M M A A V Q G K D C A R A G S A E I N F E GN E F A Y 1739 GS D FAY 1685 GT D L A Y 1652 GS N V S Y 1364 128 alpha 1A F Y F V S F I F L CS F L ML N L F V A V I MD N F E Y L T R D S S I L G P H H 1779 alpha 1B F Y F V S F F L C S F L M L N L F V A V I MD N F E Y L T R D S S I L GP H H 1725 alp ha 1E V Y F V S F I F FC S F L M L N L F V A V I MD N F E Y L T R D S S I L G P H H 1692 UNC-2 A Y F T S FV F L S S F L M L N L F V A V I MD N F D Y L T R D S S I L G P H H 1404 alpha 1A L D E Y V R V W A E Y D P A A C G R I H Y K D M Y S L L R V I S P P L G L G K K 1819 alpha 1B L D E F I R V W A E Y D P A A C G R I S Y N D M F E M L K H M S P P L G L G K K 1765 alp ha 1E L D E F V R V W A E Y D R A A C G R I H Y T E M Y E M L T L M S P P L G L G K R 1732 U N C - 2 L D E F I R V W A D Y D P A A T G R I H Y S E M Y E M L R I M A P P V G F G K K 1444 alpha 1A C P H R V A C K R L L R M D L P V A D - D N T V H F N S T L M A L I R T A L D I 1858 alpha 1B C P A R V A Y K R L V R M N M P I S N E D M T V H F T S T L M A L I R T A L E I 1805 alp ha 1E C P S K V A Y K R L V L M N M P V A E - D M T V H F T S T L M A L I R T A L D I 1771 U N C - 2 C P Y R L A Y K H L I R M N M P V A E - D G T V H F T T T L F A L I R E S L S I 1483 alpha 1A K I A K G GA D K Q Q M D A E L R K E M M A I W P N L S Q K T L D L L V T P H K 1898 alpha 1B K L A P A G T K Q H Q C D A E L R K E I S S V W A N L P Q K T L D L L V P P H K 1845 alp ha 1E K I A K G GA D R Q Q L D S E L Q K E T L A I W P H L S Q K M L D L L V P M P K 1811 U N C - 2 K M R P - V E E M D E A D E E L R L T L K K I W P L K A K K N M V D L V V P P N 1522 alpha 1A - - - - S T D L T V G K I Y A A M M M E Y Y R Q S K A K K L Q A M R E - . . . 1930 alpha 1B P D E M T V G K V Y A A L M I F D F Y K Q N K T T R D Q T H Q A P G G L 1881 alp ha 1E - - - - A S D L T V G K I Y A A M M I M D Y Y K Q S K V K K Q R Q Q L E - . . . 1843 U N C - 2 H E L C F Q K L T V G K I Y A G L L I L E N Y R A R K S G T E V G G Q G L F G G 1562 a l p h a l A E Q N R T P L M F Q R M E P P S P T Q E G G P S Q - N A L P S T Q L 1963 a l p h a l B S Q M G P V S L F H P L K A T L E Q T Q P A V L R G A R V F L - R Q K S A T S L 1920 alpha 1E E Q K - N A P M F Q R M E P S S L P Q E I I S N A - K A L P Y L Q Q 1875 U N C - 2 G L R S L V A A A K A A E S Q H S S H T P Q P P E E T T P I I P Q H A Q Q F S A 1602 alpha 1A D P G G G L M A Q E S S M K E S P S W V T Q R A Q E M F Q K T G T W S P E 2000 a lpha lB S N G G A I Q T Q E S G I K E S L S W G T Q R T Q D V L Y E A R A P - - - - L E 1956 alpha 1E D P V S G L S G R - S G - - - Y P S M S P L S P Q E I F Q L A C M D P A D 1908 U N C - 2 A P T M S A Q G S L Q Q M Q G T S S G G G Q R P Y S L F N S F V D T I K S G K Q 1642 alpha 1A R G P P I D M P N S Q P N S Q S V E M R E M G T D G Y 2027 alpha 1B R G H S A E I P V G Q P G A L A V D - V Q M Q N M T L R G P 1985 alpha 1E D G Q F Q E Q Q S L V V T D P S S M R R S F S T I R D K R S N S 1940 U N C - 2 D G D V T D V Q Y Q S V D Q Q H E K M N S T G R R L S D M F S K I R R G T S A D 1682 a lphalA S D S E H Y L P M E G Q T - R A A S M P R L P A E N Q R R R G R P R G N N L S T 2066 a lphalB - D G E P Q P G L E S Q G - R A A S M P R L A A E T Q P A P N - - - - 2014 alpha 1E S W L E E F S M E R S S E - N T Y K S R R R S Y H S S L R L S A H R L N S D S - 1978 U N C - 2 H N P H Q T E H L L A Q D N R S P S S P R Y R S M A R A S P P S P A E R Y G - - 1720 129 alpha 1A I S D T S P M K R S A S V L G P K A R R L D D Y S L E R V P P E E N 2100 alpha 1B A S P M K R S I S T L A P R P H G T - Q L C N T V L D R P P P S Q V 2047 alpha 1E - G H K S D T H R S G G R E R G R S K E R K H L L S P D V S R C N S E E R 2014 UNC-2 H P P R Y R T E S P P S S R S E Y Q M S I R D P I I R R N R Y N T M E H S 1757 alpha 1A Q R Y H Q R R R D R G H R T S E R S L G R Y T D V D T G L G T D L S M T 2136 alpha 1B S H H H H H R C H R R R D K K Q R S L E K G P S L S V D T E G A P S T A A G S G 2087 alpha 1E G T Q A D W E - - S P E R R Q S R S P S E G R S Q T P N R Q G T G S L S E S S I 2052 UNC-2 R S S H D P Q Y H Q D Q Q Q Q Q Q P H H Q Q H S Q H L Q H S H H K T Y Q N H N Q 1797 alpha 1A T Q S alpha 1B L P H alp ha 1E P S I UNC-2 YS R G D L P S G E G S T G C R R S D T S TP K D R D Q D R G R P K D R K H R P H H H S P I YS E R K Q E R G R S Q E R R Q P S S S S S E K Q R F Y S C V P P K P R P L L S Y S S L S Y R R E R E F R R Y Q D S T P R R S R R Q L P P - - D D S S V A E 2164 2127 2084 1828 alphalA H H H H H H H P P A P D alphalB D R F G S R E P P Q P K P S L S S - H P alpha 1E M R H T G G I S P P P D G S E G G - S P UNC-2 Q D V S E D D D P M P T A V R A R R L P - R E R Y A Q E 2183 I S P T A A L E P GP 2157 L A S Q A L E S N S A C L T E S S N S L 2123 L I S T M P T H Y E S A Y Q P S S Y N Q 1868 alphalA R P D T G R A R A R 2193 alphalB H P Q G S G S V N G S P L M S T S G A S T P G R G G R R Q L P Q T P L T P R P S 2197 alpha 1E H P Q Q G Q H P S P Q 2134 UNC-2 H L N D S Y G L G T G Y Q R D Y H T S H S H S H H P T S Q 1897 alphalA E - Q R W S R S P S E 2203 alpha 1B I T Y K T A N S S P V H F A E G Q S G L P A F S P G R L S R G L S E H N A L L Q 2237 alpha 1E HY I S E P Y L A L H E D S H A S D C G E E 2156 UNC-2 Q Q Q H Q P M Y S T S P L I S P R S S H S Y Y T P 1922 alp ha 1A alpha 1B alp ha 1E UNC-2 K E P L S Q P L A S G S R G S D P Y L G Q R L D S E A S A H N L P E D T L T F E T L T F R S S Q Y Y 2203 2277 2161 1928 alpha 1A G R E 2206 alphalB E E A V A T N S G R S S R T S Y V S S L T S Q S H P L R R V P N G - - Y H C T L 2315 alpha 1E E A A V A TS L G R S N T I GS AP P L R H S W Q M P NGH Y R R R R L G G L G 2201 UNC-2 E I P S P S P D I Y P S Y R G S A S P R R Y P T S T V V V A P D R E G S S A R V 1968 alphalA H A T H RQ alphalB G L S T G V R A R H S Y H H P D Q D H W C \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 alpha 1E L A M M C G A V S D L L S D T E E D D K C UNC-2 I QA QP G S I P L S D S E T E D D P RWA I V 2212 2336 2222 1992 130 Figure 13. Alignment of regions of UNC-2 with the analogous regions of the mammalian HVA VGCC. A) The II-III loop of the HVA rat brain channels, and (B) the regions of the rat brain Class C and D, human Class F, and C. elegans EGL-19 implicated in DHP binding were aligned with the analogous regions of the UNC-2 amino acid sequence using the Mac Vector Alignment Program. Alignments were imported into the SeqVu 1.1 Program. Regions of homology are shaded and regions of identity are boxed. The hatched bars in figure B underlie regions of L-type channels shown to be responsible for the DHP sensitivity (see Zhang et al, 1995) and the asterisks indicate the five amino acids critical for DHP binding by Schuster et al, 1996. 131 ALAJ OA L L LEJQ AO LEJVLAI L L L L L L L T V F 0 L T G E DWN E VHY D K S Q G G V Q G G M V F T V F 0 L T G E DWN A V M Y H G 1 E S Q G G V S K G _ M F S T V F 0 L T G E DWN E vii v N G 1 R -S. Q G G V S S G - M W S T V F Q L T G E DWN S V M Y D G 1 M A Y G G p S F P G M L V T V F 0 L T G E DWN A V M Y D G 1 M A Y G G p S S S G M | V T V F Q L T G E DWN V V M Y D G 1 M -A. Y G G p F F P G M L V T V F 0 L T G E DWN T V M Y H LG, I E S F G G V G T L G V 1 V T V F o 1 T G F DWN F V M Y L AU E -\u00C2\u00A3 Q 1 Y S G G W P Y 5 Y F Y F Y F Y F Y F YLX] Y_E_ V L V L I 699 693 637 736 735 730 686 697 IIS6 alpha 1A T L F G N Y T L L N V F L A I A V D N L A N A 0 E L T K alpha 1B T L F G N Y T L L N V F L A I A V D N L A N A Q E L T K alpha 1E T L F G N Y T L L N V F L A A V D N L A N A Q E L T K alpha 1C F I C G N Y I L L N V F L A A V D N L A D A E S L _L S alpha 1D F I C G U Y I L L N V F L A A V O N L A D A E S I N T alpha 1F F I C G N Y I L L N V F L A A V D N L A S Ja D A T A EGL-19 F I C G N Y I L L N V F L A A V D N L A D A D s L T N UNC-2 V L F fiNY T I I N V F I A A V O N I A N A Q E _L _L A E| Qi E DLEJ QI A Q K A Q KLEjE E E E E G G A N 0 K L A L Q 743 A N Q K 1 A L Q 737 F N Q K H A L Q 681 K E R K - - \u00E2\u0080\u00A2 - 776 K E R J\u00C2\u00A3 - - - 775 K S 766 717 N 734 alpha 1A alpha 1B alpha 1E alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 K A K E V A E V S P L S A A N M S I A V K E Q Q K N Q K P A K S V W E Q R T S E M R K Q K A K E V A E V S P M S A A N I S I A A R Q Q - - N S A K A R S V W E Q R A S Q L R L Q K A K E V S P M S A P N M P S I E R D R R R R H H M S M W E P R S S H L R E R K L A R P A R T A S P E K K I A R K E S L E - N K K N E K E E S E E L D E Q 787 779 720 789 787 769 717 746 alpha 1A N L L A S R E A L Y G - \u00E2\u0080\u00A2 D A A E R W P T T Y A R P L R P D V K T H L D R P L V V D P Q 829 alpha 1B N L R A S C E A L Y S E M D P E E R L R Y A S T R H V R P D M K T H M D R P L V V E P G 823 alpha 1E R R R H H M S V W E Q R T S Q L R R H M Q M S S Q E A L N K E E A P P M N P L N P L N P 764 alpha 1C K Q E V M E 795 alpha 1D N N 789 alpha 1F D 770 EGL-19 717 UNC-2 Y Q 748 alpha 1A alpha 1B alpha 1E alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 E N - R N N N T N K S R \u00E2\u0080\u009E . A P E A L R Q T A R P R E S A R D P D A R R R D G L R G P A G N K S K P E G T E A T E G A D P P R R H H R H R D R D K L S P L N P L N A H P s L Y R R P R P I E G L A L G L G L E K C E E E R I S A P E P -S R G G S L K 869 866 808 795 789 770 717 748 alpha 1A alpha 1B alpha 1E alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 R A P G R E G P Y G R E S E P O Q R E H A P P R E H V P W D A D P E R A K A G D A - A G G W- \u00E2\u0080\u00A2 - Q D R T D C P K A E S T E T G A R E E R A R - -G D I G G L T S V L D N - - - Q R S P L S L G K R E P P W L P R S C H G N - -R R LEJ R R I D P P p LEJ A V E V E G D 913 895 845 799 793 772 717 751 alpha 1A alpha 1B alpha 1E alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 - - H T H R P V A E G E P R R H R A R R R P - - GDE S H S K E A P G A D T Q V R C E R S R R H H R R G S -- T Q Q E T G G G E T V V T F E D R A R H R - - - Q -DDR E E A E R R P E R E P Q R R S E - - S Q I A R P R D A T R P 953 R R H R A H R H 938 R H R R V R T E 880 K E E K I E L K 810 S D N K V T I D 806 Q E N E G L V P 780 E E E Q Q E I E 725 H C T I D M E 758 132 alpha 1A alpha 1B alpha 1E alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 R A Q D K E I T Y Q V E E D K T G[i]G D D KLEJG K E K E G T A P V L V P K G R S R S A S Q E R S L D E G V s I D G R R H R R R K R R A R H R -K E H E - - -E S Q G S G V P M - G S' P P l l i -D K - - -E G - \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 F LEE G - - -D M C A V A R 997 968 914 821 815 789 734 770 alpha 1A alpha 1B alpha 1E alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 s G P N - - L S T T R P I Q Q D L G R Q D L P L A E D L D N M K N N K L A T G E P A S P T G P R E T E N S E E P T R R H R A K H K V P P T L E P P E R E V A E K E S N V V E G D S S H R - - K E P T I H E E E R T Q D L R R T N S L M V P R G S \u00E2\u0080\u00A2 - G L V G A L D E 1039 1012 953 821 815 789 734 770 alpha 1A alpha 1B alpha 1E alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 H D S L G H S G L P P - S P A K I G N S T N P G P A L A T N P Q N A A S R R T P N N P G K E T R N H Q P K E P R C D L E A I A V T G V G S L H M L P S T C L Q K V D E Q P E A E T P L V Q P Q P E L E V G K D A A L T E Q E A E G S S E Q A L L A D V Q L D V G R G T K I N M D D L 0 P S E N E D K S P H S N P D P Y P P C D V P V G E E E E A R R E G A D M E E E E E E E E D E G E E A M D D L 843 830 804 740 775 alpha 1A alpha 1B alpha 1E alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 D A D N Q R N p S S T 0 P N S A K T A R K P E H M A V E I P P A M G s Q P S D P S T T \u00E2\u0080\u00A2 V H V P V T L T L S c M T T N M D K A T T E S T S V T V A I P D V 1126 1083 1029 843 830 804 740 775 alpha 1A alpha 1B alpha 1E alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 alpha 1A C P P L N H T V V Q V N K N A N P D P L P K K E E E K K E E E E alpha 1B G P P G E A T V V P S - - - A N T D - L E G Q A E G K K E A E A alpha 1E D P L V D S T V V N I S N K T D G E A S P L K E A E T K E E E E alpha 1C N T A G E E D E E E P E M P V G P R P R P L S E L H L alpha 1D - E E E E D E P E V P A G P R P R R I S E L N M alpha 1F - E E E E E E E E E E G A G G V E L L Q E V V P EGL-19 H G M D E P E G D E E M T S A R P R R M S E V P A UNC-2 D E E C E E .E E S P K[P|M[I] P RLEJ I v P D G P R G P E T G KJLMJLP E K A V E K I A E K V V S T V K P P P i LED G G P KLEJM V A D P G E D D V L R E V E K K K Q K K E K R K \u00C2\u00A7\u00C2\u00A7 \u00E2\u0080\u00A2 A F IIIS1 s s s s M M SRJM G| SLA, F A IS S| Ll F J I L L S|H Is SIMIFIFI I s ip RLUV LLU) L R V_LM R Y F Y F L R l X F D T iLtsll H H L I J i H HJjJF T N L JLVLNIH S[Y F T N A \u00E2\u0080\u00A2 V c T KIV F I F M M E M C E M V E M C T N L T N L M V V V L V F F V F F C \u00E2\u0080\u00A2arc V M T VLUC L A M A L A A L L M L I L L V 1163 1116 1073 871 854 828 766 786 1207 1160 1117 915 898 872 810 830 alpha 1A S S I A L A A E D P V Q P N A P R N N alpha 1B S S I A L A A E D P V R T D S F R N N alpha 1E S S I A L A A E D P V L T N S E R N K alpha 1C S S I S L A A E D P Q H T s F R N H alpha 1D S S A A L A A E D P I R S H s F R N T alpha 1F S S V S L A A E D P I R A H s F R N H EGL-19 S S A M L A A E D P LL Q A N .a T R N M UNC-2 fi. fi V fi I A A F n P V D E E N P R N K VLUQ LM]D Y JL D .YJMLQ. V F V F GLVLF sn F A S J J F T V V F J J v FIA M V M V M V I I I L I L I T M L K M K M K M K M K M T L LLKJ v T| v LLJD QLGJ 1251 1204 1161 959 942 916 854 874 133 B alpha 1C D S S K Q T E A E C K G N Y I T Y K DG E V D H P I I Q P R S W E N S K 1098 alpha 1D D E A K S N P E E C RG L F I L Y K DG D V D S P V V R E R I WQ N S D 1081 alpha 1F D E A K H T P Q E C KG S F L V Y P DG D V S R P L V R E R L WV N S D 1055 EGL-19 D L S K M T E A E C R G E Y I H Y E DG D P T K P V S K K R V WS N N D 993 UNC-2 D K N R K F A N T C HG Q F F V Y D N Q N - D P P R V E Q R E WR L R P 1018 V W W W W M alpha 1C F D F D N V L A A M M A L F T V S T F E G WP E L L Y R S I D S H T E D 1134 alpha 1D F N F D N V L S A M M A L F T V S T F E G WP A L L Y K A I D S N G E N 1117 alpha 1F F N F D N V L S A M M A L F T V S T F E G WP A L L Y K A I D A Y A E D 1091 EGL-19 F N F D N V G D AM I S L F V V S T F E G WP Q L L Y V A I D S N E E D 1029 UNC-2 F N Y D N T I N A M L T L F V V T T G E G WP G I RQ N S M D T T F E D 1054 IIIS6 alpha 1C K G P I Y N Y R V E I S I F F I I Y I I I I A F F M M N I F V G F V I V 1170 alpha 1D V G P V Y N Y R V E I S I F F I I Y I I N V A F F M M N I F V G F V I V 1153 alpha 1F H G P I Y N Y R V E I S V F F I V Y I I I I A F F M M N I F V G F V I I 1127 EGL-19 K G P I H N S R Q A V A L F F I A F I I V I A F F M M N I F V G F V I V 1065 UNC-2 Q G P S P F F R V E V A L F Y V M F F I V F P F F F V N I F V A L I I I 1090 alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 T F Q E Q G E Q E Y K N C E L D K N Q R Q C V E Y A L K A R P L R R Y I 1206 T F Q E Q G E K E T F R A Q G E Q E T F Q N E G E R E T F Q E Q G E A E Y K N C E L Y Q N C E L Y E N C E L L S E G D L D K N Q R Q C V E Y A L K A R P L R R Y I 1189 D K N Q R Q C V E Y A L K A Q P L R R Y I 1163 D K N Q R K C I E F A L K A K P H R R Y I 1101 D K N Q K Q C I D F A L N A R P R S L F M 1126 alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 T G E A W Q D I M L A C M P G K E A WQ E E A WQ E E A WQ D E G WQ D alpha 1C C G S S F A alpha 1D C G S N F A alpha 1F C G S N F A EGL-19 C G N N F A UNC-2 C G S N V S K C A P E S E P S N S T K G - E T P 1449 M L A C M L A S M L S C L P G K L P G N S D R E M M A A V Q G K -- - L C D P D S D - - Y N P G E - E Y T - - R C D P E S D - - F G P G E - E F T D V R C D P M S D D Y H K G G L N E S R D C A R A G S A E I N F E K - G Q T IVS6 F Y F I V Y A Y F I F I F I F T S F Y M L S F Y M L S F F M L S F F M L S F V F L C A F C A F C A F C S F S S F I I I I I I V I M L N L N L N L N L N L V A V V A V V A V V A V V A V M D N M D N M D N M D N M D N D Y L D Y L D Y L D Y L D Y L 1434 1400 1335 1356 1485 1470 1436 1371 1392 alpha 1C alpha 1D alpha 1F EGL-19 UNC-2 T R D WS R D WS R D WS R D WS T R D S S L G P L G P L G P L G P L G P H H L H H L H H L H H L H H L D E F D E F D E F E E F D E F K R K R E E WA WS K R I WS V R L WS E Y I R V W A D Y Y D P E A K G R Y D P E A K G R E Y D P G A K G R D P D A K G R D P A A T G R K H L K H L K H L K H L H Y S D V V D V V D V V D V V E M Y 1521 1506 1472 1407 1428 134 contain one less positive charge than the homologous domains in their vertebrate counterparts (Figure 12). UNC-2 expressed throughout development in C. elegans. To examine the expression of unc-2, RNA was prepared from staged populations (Ll - L2, L3, and L4 - adult) and probed with the CEC-32 fragment. The probe hybridized to a single 7.5-kb band present at each developmental stage (Figure 14). These results indicate that the unc-2 gene is expressed throughout development in C. elegans and is predominantly transcribed as a ~7.5-kb transcript. In order to examine the distribution of UNC-2 in C. elegans, we generated polyclonal antibodies against bacterial fusion proteins. Two regions, the intracellular linker between domains II and III, and the carboxyl terminus, were expressed as fusions with GST. However, only the former was soluble and could be purified in sufficient quantities for injection. The corresponding sera, designated FP3 and FP4, specifically recognized the fusion protein in Western blotting experiments (data not shown). However, I was not able to obtain reproducible staining results in embryos or adult worms using these sera, nor could I detect a band of the expected size on Western blots of worm proteins. 135 Figure 14. Northern blot of staged C. elegans total RNA. Staged C. elegans total RNA was probed with the CEC-32 fragment which includes sequence encoding domain JJIS5 to domain IVS5. Analysis indicates that an ~7.5-kb message is expressed throughout development. Lane 1) L l - L2, Lane 2) L3, and Lane 3) L4 - Adult. i 3 6 origin 7.5 Kb 28 S 18 S Chapter 4. Genetic and phenotvpic analysis of unc-2 alleles. Background. To identify regions of the a, subunit that are important for proper channel function, I took advantage of C. elegans as a genetic system to generate mutations in a VGCC gene. As described in Chapter 3,1 identified a VGCC gene in C. elegans that was later shown to correspond to the unc-2 locus (Schafer and Kenyon, 1995). The unc-2 mutant phenotype was first described by S. Brenner (1974), who isolated three alleles of unc-2 (e55, e97, and el29) in screens for behavioral and morphological mutants, unc-2 mutants are sluggish and kinked, unlike wild-type (N2) animals, which move in a smooth sinusoidal wave. In addition, unc-2 mutants lay eggs in clumps, suggesting that the mutation results in unregulated egg laying. In addition, these mutants fail to adapt to the presence of exogenous dopamine and serotonin (Schafer and Kenyon, 1995) and are resistant to the acetylcholinesterase (AChE) inhibitor aldicarb (Yook and Jorgensen, personal communication; Miller et al., 1996). In this study, I isolated eleven new alleles of unc-2 using a pre-complementation screen and obtained six additional alleles that had been isolated in the Jorgensen and Rand laboratories. I then identified the underlying sequence alterations in eight of these alleles, including two missense alleles, ra611 and ra612. I examined the locomotory and 138 defecation defects in a subset of these mutants (ra605, ra611, and ra612). In addition, the sensitivity of these mutants to the AChE inhibitor aldicarb and ACh receptor agonist nicotine were tested. The missense mutations were introduced into mammalian a, subunit clones and expressed in tissue culture cells. The ra612 mutation was found to dramatically alter the activation and inactivation properties of the surrogate a, subunit. This finding illustrates the strength of a genetic approach in dissecting structure-function relationships in VGCCs. Results. Isolation of new mutations in the unc-2 gene. A pre-complementation screen was used to efficiently isolate new alleles of unc-2 (Figure 15). N2 males were mutagenized with 0.5 mM EMS and crossed to dpy-3(e27) unc-2(e55) (DM2601) hermaphrodites. The dpy-3 locus is less than 1.5 map units from unc-2 and the e27 mutation results in a recessive \"dumpy\" phenotype (Brenner, 1974). Two major classes of outcross progeny were expected: Wild-type hermaphrodites (dpy-3 unc-2/+ +) and Dpy Unc males (dpy-2 unc-2/O). However, if new unc-2 mutations are induced, these matings will also give rise to rare Unc non-Dpy hermaphrodites (dpy-3 unc-2/+ unc-2(new)). Twenty mating plates, each with six males and three hermaphrodites, were set up, and the F, progeny screened for the presence of Unc non-Dpy progeny. Eleven independent Unc non-Dpy animals were identified and picked to new plates. These animals each gave rise to the expected 3:1 ratio of Unc to Dpy Unc progeny. From each plate, single Unc animals were transferred to new plates and their progeny were scored for the presence of Dpy Uncs. Animals that failed to segregate Dpy Unc progeny were presumed to be homozygous for the new unc-2 mutation (unc-2(new)/unc-2(new)). 139 Figure 15. Flowchart illustrating the precomplementation screen used to isolate novel alleles of unc-2. Wild-type males were mutagenized with 0.5 mM EMS and allowed to mate with hermaphrodite animals homozygous for unc-2(e55)dpy-3(e27). F l hermaphrodite progeny were screened for the presence of an uncoordinated, non-dumpy phenotype. Animals homozygous for the new unc-2 allele were selected from the F2 generation. 140 -f- 11 P o : Phenotype: Genotype: wt + + Mutagen X dpy unc unc-2 dpy-3 unc-2 dpy-3 Phenotype: dpy unc W t dpy unc unc-2 dpy imc dpy-3 Genotype:- unc-2 dpy-3 unc-2 dpy-3 unc-2 dpy-3 unc-2 dpy-3 unc-2 dpy-3 unc-2 dpy-3 0 + ;+' . 0 unc-2* + 0 + dpy-3 F2: Phenotype: Genotype: discard pick pick dpy unc unc-2 dpy-3 unc-2 dpy-3 unc-2 unc-2 dpy-3 unc-2* + unc-r2 unc-2* + unc-2* + F3: segregates dpyuncs discard only segregates unc-2 retain Key: * = new allele + = wildtype ( ^ ) = self-segregate A : neither unc-2 or dpy-3 are hit B : unc-2 gene hit C: dpy-3 gene hit 141 To ensure that these new unc-2 strains did not carry mutations in other genes, the animals were outcrossed, and the homozygous mutants were re-isolated from the heterozygous progeny. Strains were outcrossed at least three times before phenotypic analysis. Eleven new alleles of unc-2 were recovered from a screen in which approximately 20,000 Fl progeny were examined. These alleles, as well as those isolated in the Jorgensen (personal communication) and Rand (Miller etal., 1996) laboratories were categorized based on a qualitative evaluation of the severity of their locomotory defects (Table 6). For example, animals were ranked as \"mild\", \"moderately severe\", or \"severe\" Uncs based on a subjective assessment of the tendency to move spontaneously and the distance, speed and time spent in motion in response to tapping the plate or gentle prodding. In addition, there was variation in the severity of the phenotype amongst individual strains of unc-2. Although all alleles exhibited the defects described above, some alleles are more severe than others. It is important to note, however, that even the \"severe\" Unc-2 phenotype is relatively mild compared to other Unc mutations. Alleles ra603, ra605, ra606, ra610, ra6U, ra613, md328, mdll86, ox2, e97, el29, and e2379 exhibited relatively severe movement deficits, while ra607, ra608, and ra609 had moderately severe deficits. Finally, alleles ra612, mdl07, mdl064, and ox8 are only mildly uncoordinated. These differences in severity suggest that the underlying nature of the mutations differ. I have analyzed these unc-2 alleles and identified the sequence changes responsible for eight of the mutant phenotypes (Table 7). Three mutations result in putative null alleles of unc-2. Five of the unc-2 mutations (ra605, ra610, ra611, ra612, and md328) were identified using an RNase Protection Assay. In this procedure, described in Methods and Materials and Figure 3, single-stranded RNA prepared from fragments of unc-2 genomic DNA was annealed to the corresponding fragment amplified from the N2 genome. The double-stranded RNA hybrids were digested with various RNases and electrophoresed. Regions 142 Table 6: Mutant alleles of unc-2. Allele Mutagen Phenotype Reference e55 EMS mod. severe Unc Brenner (1974). e97 EMS mod. severe Unc Brenner (1974). el29 EMS mod. severe Unc Brenner (1974). e2379 EMS mod. severe Unc Thierry-Mieg ra603 EMS Severe Unc * ra604 EMS Mild Unc * ra605 EMS Severe Unc * ra606 EMS Severe Unc * ra607 EMS mod. severe Unc * ra608 EMS mod. severe Unc * ra609 EMS mod. severe Unc * ra610 EMS Severe Unc * ra611 EMS Severe Unc * ra612 EMS Mild Unc * ra613 EMS Severe Unc mdl07 Spontaneous Mild Unc Miller etal. (1996) md328 Spontaneous Severe Unc Miller etal. (1996) mdl064 Spontaneous Mild Unc Miller etal. (1996) mdll86 Spontaneous Severe Unc Miller etal. (1996) ox2 EMS Severe K. Yook and E. Jorgensen 0x8 EMS Mild K. Yook and E. Jorgensen ra614 Spontaneous Wild-type * allele was isolated in this study Table 7. Molecular alterations in unc-2. Allele Mutation Sequence alteration Region ra605 C ^ T 01252-4 stop IVS4 ra610 C->T R1343-> stop rvs5-rvs6 ra611 G->A G1254^ R IVS4 ra612 G->A G1442-> R carboxyl tail md328 deletion/insertion/ duplication deletes splice acceptor sequence IIS6 mdll86 Tel insertion S138-Tcl-Y139 IS2-IS3 mdl064 Tel insertion Y1255-Tcl-T1256 IVS4-IVS5 ra614 A->G Y1255C rVS4-TVS5 143 of mismatch are digested by the RNases, resulting in a band shift that can be detected on an agarose gel containing ethidium bromide (Figure 16). Fragments identified as containing mutations were subsequently analyzed by Cycle Sequencing. Two of these mutations, ra605 and ra610, are nonsense mutations. In ra605, a C to T transition alters glutamine 1253 to a stop codon. Likewise, in ra610 the CGA encoding arginine 1344 is altered to a TGA stop codon. These two mutations truncate the a, protein in the IVS4 segment and the domain IV P-loop, respectively. Allele md328 is a complex rearrangement in intron 12 and exon 13 that destroys the splice acceptor consensus sequence. This mutation is predicted to result in the deletion of the latter part of segment US6 and introduce a premature termination codon. Since previous studies have demonstrated that truncations of the a, subunit at the proximal portion of the carboxyl terminus result in non-functional proteins (A. Stea, personal communication; Wei et al, 1994), these three mutations probably represent the null state of the unc-2 gene. The locations of these mutations are shown in Figure 17. Two unc-2 mutations are single amino acid substitutions. Two alleles, ra611 and ra612, are missense mutations that result in the substitution of an arginine residue for a glycine (Figure 17). The ra611 allele is a G to A transition in IVS4 that converts glycine 1254 to an arginine. The ra612 allele is a G to A transition in the carboxyl terminal region, approximately 63 residues after IVS6, that also alters a glycine to an arginine (G1442R). The glycine residues altered in the ra611 and ra612 mutants are conserved in all vertebrate HVA VGCCs cloned to date (see Stea et al, 1995a). Two alleles of unc-2 are transposon-induced. C. elegans contains a family of transposable elements (transposons, Tc) (Emmons, 1988; reviewed in Moerman and Waterston, 1989). Transposable elements are mobile, meaning that an element can excise from one chromosomal position and insert at a new site in the 144 Figure 16. Typical example of positive results from RPA screen. A photograph of a 2.0% agarose gel on which ds-RNA fragments were electrophoresed after RNase digestion. The ra612:Tl+ N2:SP6 ds-RNA fragment contains a mismatch that is digested by the RNase, resulting in a lower molecular weight product (indicated by the arrow). See details in Methods and Materials. 145 mm mm mm m m 146 Figure 17. Location of mutations in unc-2. Transmembrane segments are indicated with bars above the sequence and conserved motifs with bars below. The locations of the mutant alleles are indicated by arrows: Tel elements (alleles mdll86 and mdl064) are inserted between the amino acids highlighted in bold, the sequence of the md328 allele is disrupted at the residue indicated with the asterisk, and the amino acid altered in ra605, ra610, ra611, and ra612 are indicated by an arrow. 147 M I K E A V Q M A V W P A L P R L A A E E A R R E Q K A E S G T F W K T T L S S N A P V K E K G P S S L F I F A E D N 60 I S 1 I S 2 I I R R N A K A I I E W G P F E Y F I L L T I I G N C W L S M E Q H L P K N D K K A L S E W L E R T E P Y F M G I F C 120 ( I S 2 ) I S 3 L E C V L K V I A F G F A L H K G S Y L R S G W N I M D F I V W S G W T M L P F S P A T Q T A N Q P V D S v T J L R T 180 \u00E2\u0080\u00A2L> mdll86 IS4 IS5 L R A V R V L R P L K L V S G I P S L Q W L K S I L C A M A P L L Q I G L L V L F A I I I F A I I G L E F Y S G A F H 2 4 0 S A C Y N E R G E I E N V S E R P M P C T N K T S P M G V Y N C D V K G T T C L Q K W I G P N Y G I T S F D N I G F A M 3 0 0 I S 6 I T V F Q C I T M E G W T T V M Y Y T N D S L G S T Y N W A Y F I P L I V L G S F F M L N L V L G V L S G E F A K E R E 3 60 R V E N R R E F L K L R R O O O I E R E L N G Y L E W I L T A E E V I L K E D R T T E E E K A A I M E A R R R A A N K K 42 0 P s u b u n i t b i n d i n g r e g i o n L K Q A S K Q Q S T E T E E D F E E D E D E M E E E Y V D E G G T V E D E F A E R K K R G C C H S V G K F I K Q L R I Q 4 8 0 I I S 1 IIS2 I R I M V K T Q I F Y W S V I T L V F L N T C C V A S E H Y G Q P Q W F T D F L K Y A E F V F L G I F W E M L L K L F 540 I I S 3 I I S 4 A M G S R T Y F A S K F N R F D C W I V G S A A E V I W A E W G G S F G I S V M R A L R L L R I F K L T S Y W V S L 600 I I S 5 R N L V R S L M N S M R S I I S L L F L L F L F I L I F A L L G M Q L F G G R F N F P T M H P Y T H F D T F P V A L I T 660 * md328 I I S 6 V F Q I L T G E D W N E V M Y L A I E S Q G G I Y S G G W P Y S I Y F I V L V L F G N Y T L L N V F L A I A V D N L A N 720 A Q E L T A A E E A D E K A N E I E E E S E E L D E Q Y Q E G D H C T I D M E G K T A G D M C A V A R A M D D L D E E C 7 8 0 I I I S 1 E E E E S P F G G P K P M V P Y S S M F F L S P T N P F R V L I H S I V C T K Y F E M M V M T V I C L S S V S L A A E D 8 4 0 I I I S 2 I I I S 3 P V D E E N P R N K V L Q Y M D Y C F T G V F A C E M L L K L I D Q G I L L H P G S Y C R D F W N I L D G I W T C A L 9 0 0 I I I S 4 F A F G F A G T E G S A G K N L N T I K S L R V L R V L R P L K T I K R I P K L K A V F D C W N S L K N V F N I L I V 9 6 0 I I I S 5 Y F L F Q F I F A V I A V Q L F N G K F F F C T D K N R K F A N T C H G Q F F V Y D N Q N D P P R V E Q R E W R L R P F 1 0 2 0 I I I S 6 N Y D N T I N A M L T L F W T T G E G W P G I R Q N S M D T T F E D Q G P S P F F R V E V A L F Y V M F F I V F P F F 1 0 8 0 ( I I I S 6 ) F V N I F V A L I I I T F Q E Q G E A E L S E G D L D K N Q K Q C I D F A L N A R P R S L F M P E D K N S T K Y R I W R 1 1 4 0 148 I V S 1 I V S 2 L V T S P P F E Y F I M T M I C C N T L I L M M K Y Y N N P L F Y E E I L R L F N T A L T A V F T V E S I L K I L A F G 12 00 ra605 T V S 3 T V S 4 I r*ra614 V R N Y F R D G W N R F D F V T W G S I T D A L V T E F G G H F V S L G F L R L F R A A R L I R L L Q Q G Y T I R I L 1 2 6 0 T ftmdl064 I V S 5 ra611 L W T F V Q S F K A L P Y V C L L I G M L F F I Y A I V G M Q V F G N I W L N A A T E I N R H N N F Q S F F N A V I L L 1 3 2 0 ra610 4 I V S 6 F R C A T G E G W Q D I M M A A V Q G K D C A R A G S A E I N F E K G Q T C G S N V S Y A Y F T S F V F L S S F L M L N 13 80 L F V A V I M D N F D Y L T R D S S I L G P H H L D E F I R V W A D Y D P A A T G R I H Y S E M Y E M L R I M A P P V G 1 4 4 0 ra612 E F h a n d m o t i f i F G K K C P Y R L A Y K H L I R M N M P V A E D G T V H F T T T L F A L I R E S L S I K M R P V E E M D E A D E E L R L 1 5 0 0 T L K K I W P L K A K K N M V D L W P P N H E L C F Q K L T V G K I Y A G L L I L E N Y R A R K S G T E V G G Q G L F 1 5 6 0 GGGLRSLVAAAKAAESQHSSHTPQPPEETTPIIPQHAQQFSAAPTMSAQGSLQQMQGTSS 162 0 G G G Q R P Y S L F N S F V D T I K S G K Q D G D V T D V Q Y Q S V D Q Q H E K M N S T G R R L S D M F S K I R R G T S 1 6 8 0 A D H N P H Q T E H L L A Q D N R S P S S P R Y R S M A R A S P P S P A E R Y G H P P R Y R T E S P P S S R S E Y Q M S 1 7 4 0 I R D P I I R R N R Y N T M E H S R S S H D P Q Y H Q D Q Q Q Q Q Q P H H Q Q H S Q H L Q H S H H K T Y Q N H N Q Y S R 1 8 0 0 S P I Y S D D S S V A E S Y R R E R E F R R Y Q D S T P Q D V S E D D D P M P T A V R A R R L P L I S T M P T H Y E S A 1 8 6 0 Y Q P S S Y N Q H L N D S Y G L G T G Y Q R D Y H T S H S H S H H P T S Q Q Q Q H Q P M Y S T S P L I S P R S S H S Y Y 1 9 2 0 T P R S S Q Y Y E I P S P S P D I Y P S Y R G S A S P R R Y P T S T V W A P D R E G S S A R V I Q A Q P G S I P L S D 1 9 8 0 S E T E D D P R W A I V 1992 149 genome. Transposon insertion into a gene often disrupts gene function, resulting in a mutant phenotype. Two members of the Tc family, Tel and Tc3, have been shown to be active in the germ line and create heritable mutations. Transposons have been used to isolate new mutations in a number of genes and to subsequently map and clone these genes (Greenwald, 1985; Moerman etai, 1986; Emmons 1988; reviewed in Moerman and Waterston, 1989). Tcl-induced mutations are typically unstable, and excision of the element occurs spontaneously in both somatic and germ line cells. Although the excision process itself is precise, exonuclease digestion and interrupted repair of the excision site often results in small deletions (Moerman et al, 1991). Alternatively, several Tel nucleotides may remain behind, creating an insertion mutation. Transposition events that occur in the germ line will be inherited by the progeny and result in new alleles of the targeted gene. Thus, transposon insertions in a gene can provide a powerful means by which to generate new alleles of that gene. Furthermore, the nature of the mutations resulting from Tel excision can be quickly and easily identified because the site of the initial Tel insertion is known. Two of the unc-2 alleles provided by the Rand laboratory (Miller et al., 1996), mdl064 and mdll86, were isolated in a mutator background, and were likely to be the result of Tel insertions in unc-2. Both alleles revert spontaneously at a high frequency (> lxlO\"4: E. A. Mathews, this study), supporting this hypothesis. A PCR-based approach was used to determine whether mdl064 or md!186 were in fact the result of Tel insertions. Primer sets consisting of p618, an oligonucleotide complementary to the 3' end of Tel adjacent to the inverted repeat, and oligonucleotides derived from the unc-2 sequence were used to amplify genomic DNA isolated from the putative Tcl-induced unc-2 mutants. Because the Tel element can insert into the genome in either orientation, both sense and antisense primers were selected to cover the portion of the gene for which the sequence was known (Figure 18). 150 The results of these experiments indicate that both mdl064 and mdll86 are the result of Tel insertions in unc-2. In the case of mdl064, the p618-EM56 primer pair amplified an approximately 800-bp fragment. This result localized the site of Tel insertion to the small cytoplasmic loop connecting IVS4 and IVS5 (Table 7, Figure 17). Further PCR experiments in which EM57 or EM59 were substituted for EM56 confirmed this result (data not shown). Sequencing of the p618-EM56 PCR product indicated that the Tel is inserted at a TA dinucleotide between Y1254 and T1255 (Table 7, Figure 17). Amplification of genomic DNA isolated from mdll86 using the p618-EM83 primer set resulted in an approximately 900-bp PCR product. The EM83 primer is homologous to the non-coding strand near the 5' end of T02C5, indicating that the Tel element is inserted in the region of unc-2 contained in the gap between cosmids T02C5 and F25H10. To confirm this result, the p618-EM83 PCR product was subcloned into the pGEM-T vector and the DNA sequence was determined. Sequencing of the cloned PCR product indicates that the Tel element is inserted in the intracellular loop between IS2 and IS3 between S138 and Y139 (Table 7, Figure 17). Revertant animals from Tel-induced strains appear upon spontaneous excision of the transposable element. By visually screening plates of RM1064 and RM1186 animals, I isolated and sequenced several revertants from each of these strains. Five of the 10 revertant alleles from the RM1064 strain and 13 of 14 isolated from the RM1186 strain had reverted to the wild-type sequence. Three, two from the RM1064 strain and one from the RM1186 strain, contain silent mutations, and three RM1064-derived alleles have an A to G transition that changes tyrosine 1254 to a cysteine residue (Table 7). Since these alleles have the same sequence alteration, only one, designated ra614, was maintained and analyzed further. 151 Figure 18. Diagram illustrating the PCR strategy for localizing the Tel elements in mdl064 and mdll86. The Tel primer p618 was paired with oligos generated from unc-2 sequence. PCR was performed with individual primer sets in an attempt to amplify fragments from genomic DNA. The location of the unc-2 oligos chosen for the PCR are indicated on the gene diagram. Forward oligos are represented above and reverse oligos are below. (Scale: 8mm = 500 bp) 152 153 Interactions between unc-2 and other genes involved in Ca2+-mediated signaling. Genetic analysis provides a powerful tool for identifying interacting genes and gene products in vivo. For example, genetic screens for \"enhancer\" or \"suppressor\" mutations are frequently used to identify interacting genes in Drosophila melanogaster and C. elegans. Suppressors are second-site mutations that hide or \"suppress\" the mutant phenotype of a particular mutation (reviewed in Herman, 1988). For example, certain mutations in the unc-54 gene, which encodes myosin heavy chain A (MHC A), suppress mutations in the unc-22 gene, which encodes a myosin light chain kinase (Moerman et al., 1982; Benian et al., 1989). Conversely, enhancers are second-site mutations that worsen or \"enhance\" the phenotype of a particular mutation (reviewed in Herman, 1988). For example, mutations in the mec-8 gene enhance the phenotype of unc-52(viable) mutations, resulting in a \"synthetic lethal\" phenotype (Lundquist and Herman, 1994; Lundquist etai., 1996). In both of these examples, the genetic interactions are indicative of specific functional relationships. In this study, I took advantage of C. elegans as a genetic system to look at interactions between UNC-2 and other proteins involved in Ca2+-mediated signaling. To identify genes that interact genetically with unc-2,1 used two different approaches: i) I screened for suppressors of unc-2, and ii) I constructed double mutants with unc-36 and egl-19 , which encode a28 (Lobel and Horvitz, 1993) and L-type a, subunits (Lobel et ai, 1994), respectively. Screens for either dominant or recessive suppressors of unc-2(e55) and unc-2(ra612) were performed using several different strategies. In the first screen, EMS was used as the mutagen and plates were screened for animals that moved better than unc-2(e55). In excess of 1,000,000 genomes were examined, but we were unable to identify suppressed animals. In the second screen, EMS was again used as the mutagen to obtain suppressors of unc-2(ra612). I hoped that a missense mutation might be a more 154 appropriate starting point for the identification of suppressor mutations. For example, while it may not be possible to by-pass the physiological requirement for a VGCC, it might be possible to alter modulatory proteins or downstream effectors of Ca 2 + signaling to compensate for a reduction in channel function. In this screen, we again examined in excess of 1,000,000 genomes and were unable to identify suppressed animals. Finally, I repeated this screen using N-nitroso-N-ethylurea (ENU) as the mutagen. Since ENU generates AT-GC transitions and GC-CG transversions (as well as a small percent of deletion events), we speculated that it might be a more effective mutagen because it is predicted to result in more missense mutations than EMS (which frequently results in nonsense mutations) (De Stasio etal, 1994). In addition, ENU mutagenesis was expected to directly revert the ra612 mutation back to wild-type at a low frequency. In this screen, we examined in excess of 1,000,000 genomes and again were unable to identify suppressed animals. I also chose to examine genetic interactions between unc-2 and other VGCC genes by constructing double mutants. The egl-19 gene encodes a putative L-type VGCC a, subunit (Tables 3 and 5; Figure 11) (Lee et ai, 1997). Null mutations in egl-19 result in a Pat (paralyzed arrest at embryonic two-fold stage) phenotype, which is associated with severe defects in muscle assembly or function (Lee et al, 1997; reviewed in Moerman and Fire, 1997). However, there are several viable alleles, including n582, which result in slow movement and defects in egg-laying behavior (egg-laying defective). Several egl-19(n582); unc-2 double mutants were constructed, including egl-19(n582); unc-2(ra605), egl-19(n582); unc-2(ra611), and egl-19(n582); unc-2(ra612), and in each case, the double mutant was almost completely paralyzed. Similar results were reported by Schafer et al. (1996) using different unc-2 alleles. In addition, Schafer et al. (1996) reported that egl-19(n582); unc-36(e251) double mutants also exhibit a paralyzed phenotype. These results demonstrate that the UNC-2 and EGL-19 channels are both required for locomotion in C. elegans. 155 The unc-36 gene encodes a VGCC 0^ /8 subunit similar to those expressed in vertebrate nervous systems (Table 3) (Lobel and Horvitz, 1993; Wilson et al., 1994). Null mutations in unc-36 result in an uncoordinated phenotype that is very similar to that of unc-2 mutants, suggesting that unc-2 and unc-36 may encode proteins that are part of the same VGCC complex in vivo. A prediction of this hypothesis is that unc-36(null); unc-2(null) double mutants should have no worse a phenotype than either single mutant. In addition, we expected that unc-36; unc-2(ra612) double mutants should be more severe than unc-2(ra612) single mutants, but no worse than either unc-36 or unc-2 null mutants. I constructed unc-36(e251 );unc-2(ra605), unc-36(e251); unc-2(ra611), and unc-36(e251); unc-2(ra612) double mutants and found that in all cases, the double mutants were indistinguishable from either unc-36(e251) or unc-2(ra605) single mutants. The details of the phenotypic analysis are presented below (see section on Phenotypic Analysis of Mutants). These observations are consistent with the hypothesis that UNC-36 and UNC-2 function as part of the same VGCC complex. Phenotypic analysis of unc-2 alleles. Mutations in the unc-2 gene affect a wide range of behaviors, including movement (Brenner, 1974), egg-laying (Schafer and Kenyon, 1995), defecation (Miller et al., 1996), and pharyngeal pumping (Avery, 1993). In addition, there are defects in adaptation to the presence of exogenous neurotransmitters such as dopamine and serotonin (Schafer and Kenyon, 1995). For example, both wild-type and unc-2 animals become paralyzed in the presence of exogenous dopamine. However, while wild-type animals typically recover within several hours and begin to move and forage again, unc-2 animals remain paralyzed (Schafer and Kenyon, 1995). unc-2 mutants are also resistant to the effects of the AChE inhibitor aldicarb, implicating UNC-2 in cholinergic transmission (Miller etai., 1996). unc-2 is also required for a subset of neuronal cell migrations in C. elegans (T. Tarn, E. Mathews, T. P. Snutch, and W. R. Schafer, submitted). In unc-2 mutants, the 156 touch receptor neuron AVM, the interneuron SDQR, and the motor neurons VC4 and VC5 often migrate randomly, leading to misplacement of their cell bodies. However, even in mutant animals with misplaced neuronal cell bodies, the neurons undergo normal differentiation and extend axons in a wild-type pattern. Thus, the UNC-2 VGCC appeared to specifically direct migration of the neuronal cell body, but is not required for axonal pathfinding or cell specification. The motor neurons that synapse onto the body wall muscles in C. elegans release ACh, which activates nicotinic ACh receptors on the muscle and causes the muscle to depolarize and contract. Stimulation of the muscle is terminated by the breakdown of the ACh in the synaptic cleft into acetate and choline by acetylcholinesterase (AChE). Aldicarb and other AChE inhibitors prevent the breakdown of ACh in the synapse, resulting in excessive stimulation of the body wall muscles. This causes paralysis and ultimately death in wild-type C. elegans. However, some mutations confer resistance to the effects of these ACh inhibitors (Brenner, 1974; Nguyen etal, 1995; Miller et al., 1996). Resistant animals are capable of growing to adulthood and producing viable progeny in the presence of aldicarb. In addition, resistant animals do not hypercontract to the same extent as do wild-type animals. The degree of resistance conferred by a mutation is directly related to the extent of the reduction in ACh release or sensitivity. Aldicarb resistant (or ric) mutants fall into two classes; one class consists of mutations that decrease the amount of ACh release from the presynaptic cell, while the other is comprised of mutations that decrease the sensitivity of the post-synaptic cell to the neurotransmitter. These two classes of mutants can be differentiated on the basis of their sensitivity to the nicotinic ACh receptor agonists nicotine and levamisole. Mutants in the first class, such as cha-1 and unc-17, are resistant to aldicarb but retain the wild-type response to nicotine and levamisole. This indicates that the resistance is the result of a decrease in ACh release rather than a deficit in the response (Rand and Nonet, 1997). In contrast, mutations in the ric-3 gene confer 157 resistance to levamisole as well as aldicarb, indicating that the gene product acts postsynaptically in ACh reception (Miller etal., 1996). We tested the sensitivities of several wnc-2mutants {ra612, ra611, and ra605) to aldicarb and nicotine. Within one hour (at =20\u00C2\u00B0) of being placed on NGM plates containing 0.5 mM aldicarb, wild-type animals became paralyzed and the body wall muscles hypercontract, causing the eggs to be extruded from the uterus. In contrast, unc-2 animals were not noticeably affected after one hour. After a week of aldicarb exposure, the wild-type animals had died without successfully reproducing. While the unc-2 animals had become hypercontracted by this point, the original animals were alive and live progeny were present. The most noticeable differences among the three unc-2 alleles tested became apparent after three weeks of aldicarb exposure (Table 8). While all three strains produced viable progeny, there were approximately 33 to 50% more animals on the ra611 and ra605 plates than on the ra612 plates. Finally, all unc-2 strains hypercontracted in response to nicotine. Taken together, these results indicate that mutations in unc-2 reduce ACh release at the NMJ, but do not interfere with its postsynaptic reception. For a more quantitative assessment of the neurotransmission defects in unc-2, we focused on two easily studied behaviors, locomotion and defecation. These behaviors are chiefly mediated by two different neurotransmitters, ACh and GAB A (reviewed in Chalfie and White, 1988). Both of these behaviors are well-characterized in C. elegans and can be analyzed using simple, quantitative assays (Miller et al, 1996). In C. elegans, ACh is an excitatory neurotransmitter that stimulates contraction of the body wall muscles. As noted earlier, unc-2 mutants are slow-moving and are resistant to the AChE inhibitor aldicarb, suggesting that cholinergic transmission is impaired (Miller et al., 1996). We used a thrashing assay to measure the extent of this impairment in various unc-2 alleles (See Methods and Materials). Thrashing was greatly reduced in unc-2(ra605), unc-2(ra611), and unc-2(ra612) homozygous mutants compared with wild-type animals (Table 9; Figure 19). Thrashing in unc-2(ra605) and unc-2(ra611) mutants was reduced to 2.6% and 2.7%, 158 Table 8. Summary of aldicarb and nicotine resistance. Strain Mutation Aldicarb (0.5 mM) Nicotine (1%) N2 wild-type dead hypercontract unc-2(ra612) G1442-> R viable, slow growing hypercontract unc-2(ra611) G1254^R viable hypercontract unc-2(ra605) Q1253-> stop viable hypercontract n = 10 animals Table 9. Summary of the thrashing and defecation rates of mutant strains. Strain N2 Thrashing (% of wild-type) 100 Defecation (% failure) 2 unc-2(ra612) 6.5 t 44 t unc-2(ra611) 2.6 * 71 * unc-2( ra605) 2.7 * 68 * unc-2(ra614) n.d. 37 t unc-36(e251) 3.2 * 68 * unc-36; ra612 2.6 * 65 * unc-36; ra605 2.6 * 63 * exll9(n582) n.d. 53 t egl-19;ra605 n.d. 8 6 t unc-25(e265) n.d. 72* n = 10 animals t = significantly different from unc-2(ra605) * = significantly different from unc-2(ra612) n.d. = not determined 159 Figure 19. Thrashing analysis of the unc-2 alleles. The thrashing defects of the eight unc-2 alleles, unc-36(e251), and unc-36; unc-2 double mutants are shown as a percentage of the wild-type thrashing rate, which was 166.8 \u00C2\u00B1 10.5. Bars represent average of data collected from 10 animals from each strain. All mutants were significantly different from wild-type (P < 0.01, Student's t-test). The asterisks indicate strains that were significantly different from unc-2(ra612) but not from one another. 160 161 respectively, of wild-type rates measured under the same conditions. Thrashing in unc-2(ra612) mutants was also reduced, but not to the same extent as the other two alleles (6.5%); unc-2(ra612) mutants were significantly more active than either unc-2(ra605) or unc-2(ra6J 1) mutants. In addition, we found that unc-36(e251) single mutants exhibited defects in thrashing behavior (3.2% of the wild-type rate), and the thrashing rates of unc-36(e25J); unc-2(ra605) and unc-36(e251); unc-2(ra612) double mutants were reduced to 2.6% of the wild-type rate. The rates obtained for unc-36(e251) and unc-36(e251) doubles were not statistically different from those observed with unc-2(ra605) single mutants, but were lower than those of unc-2(ra612) single mutants. The results of the thrashing assay provides support for a defect in cholinergic transmission unc-2 animals. To assess the effects of mutations in unc-2 on the release of other neurotransmitters, we examined the EMC (enteric muscle contraction) step of the defecation cycle. The EMC is the last of three steps in the defecation motor program (DMP) and is mediated by the neurotransmitter GABA (Mclntire et ai, 1993[a,b]). Interestingly, in the case of the EMC, GABA acts as an excitatory amino acid; GABA released from the AVL and DVB motor neurons produces muscle contraction in the two muscles surrounding the posterior intestine, the anal depressor and sphincter muscles, resulting in expulsion. We examined several unc-2 mutants for failure in the expulsion step of the defecation cycle which is indicative of a defect in GABA neurotransmission. As in the thrashing assay, we compared unc-2(ra612), unc-2(ra611), and unc-2(ra605) with wild-type animals. The expulsion failure rates of these three unc-2 mutants were statistically different from that of wild-type (Table 9; Figure 20). Wild-type animals failed in fewer than 2% of cycles, while unc-2(ra612) mutants had an approximately 44% failure rate, and unc-2(ra605) and unc-2(ra611) mutants failed -70% of the time. This rate of failure was not significantly different from that of unc-25(e265) mutants; unc-25 encodes glutamic acid decarboxylase (Jin etai, 1999), and strong alleles of this gene, including e265, completely 162 Figure 20. Defecation analysis of the unc-2 alleles. The expulsion failure rate of eight unc-2 alleles, unc-36(e251), and unc-36; unc-2 double mutants and wild-type are shown as a percentage. The bars represent the pooled values obtained from 10 animals from each genotype observed for 10 consecutive defecation cycles. Differences observed between wild-type and mutant strains are statistically significant (P < 0.01, Student's t-test) as are differences between unc-2(ra612) and all strains (indicated by an asterisk) except egl-19(582) and egl-19(582);unc-2(ra605) and egl-J9(e582);unc-2(ra612). The \\f indicated strains with significantly different expulsion defects from unc-2(ra605). The differences between the following pairs were not significant: unc-2(ra605) and unc-36(e251), unc-2(ra605) and the unc-2 double mutants with unc-36(e251), unc-36(e251) and the unc-2 double mutants with unc-36(e251) and the unc-2(ra605), unc-2(ra611), unc-36(e251), and doubles thereof from unc-25(e265). 163 164 eliminate GAB A biosynthesis. In addition, we found that the ra605 and ra611 animals failed significantly more often than ra.612 animals. We also examined the expulsion failure rates of unc-36(e251) single mutants, and unc-36(e251); unc-2(ra605) and unc-36(e251); unc-2(ra612) double mutants. The defects observed in these animals were not significantly different than that of the unc-2(ra605) allele alone (Table 9; Figure 20). In summary, unc-2 mutants have defects in both locomotion and defecation, implicating UNC-2 in both cholinergic and GABAergic transmission. In addition, unc-2 and unc-36 single mutants, and unc-36; unc-2 double mutants were found to have similar defects in both behaviors. However, unc-2(ra612) mutants exhibited significantly milder defects in both locomotion and defecation, suggesting that this allele is not null. 165 Chapter 5. Electrophysiological analysis of altered q t subunits in a heterologous expression system. Background. To understand the relationship between protein structure and its physiological roles requires analysis of the protein at a functional level. Cloned VGCCs are often studied in heterologous systems which allow specific channel types to be studied in isolation from other ionic currents which can be difficult to eliminate using pharmacological methods. VGCC cDNAs can be injected directly into the nucleus of Xenopus oocytes or transfected into cells in culture, and transcription and translation of the transfected DNA is driven by the host machinery (Stea et al, 1993; Tomlinson etai, 1993; Soong etai, 1993; Bourinet et al, 1999). Expression of cDNA clones in Xenopus oocytes and mammalian cell lines has been widely used to study the electrophysiological and pharmacological properties of the different classes of VGCCs (Mori et al, 1991; Williams et al, 1992a; Ellinor et al, 1993; Stea et al, 1993; Tomlinson et al, 1993; Sather etai, 1993; Soong etai, 1993; Niidomi et al, 1994). In addition, these systems have been used to examine the regulation of ion channels by modulatory proteins such as kinases and G-protein, as well as assess the effects of ancillary channel subunits (a2/8, p\ and y) on the properties of the pore-forming a, subunits. 166 Results. To examine the effects of the ra611 and ra612 mutations on VGCC properties, electrophysiological analyses were conducted on HEK tsa201 cells expressing a, subunits containing the corresponding mutations. While it would be ideal to assess the effects of these mutations in the UNC-2 channel, the 5' end of the unc-2 gene has only been recently identified (E. A. Mathews, this study) and a functional clone is not yet available. Furthermore, once such a clone is constructed, a detailed characterization of its properties will need to be performed. However, since the ra611 and ra612 mutations alter residues conserved in all HVA VGCC subunits cloned to date (see review by Stea et al, 1993), it seemed reasonable to assume that the effects of the mutations are not restricted to UNC-2 channel function, but are essential to the proper function of all HVA VGCCs. Initially, both the ra611 and ra612 mutations were introduced into the N-type cx1B clone from rat brain (Dubel et al., 1992) as UNC-2 shares the greatest sequence homology with this channel class. However, low levels of expression of the oc1B.612 construct prompted us to introduce the ra612 mutation into the rat brain a!A clone (Starr et al, 1991), which is expressed more robustly in HEK tsa201 cells than the a1B (A. Stea, personal communication). The cDNA clones were co-transfected with oc25 and p subunit cDNAs into HEK cells using the Ca2+-phosphate technique. Whole cell electrophysiological properties of the mutant channels were examined and compared with those of the wild-type ct1A channels. The unc-2(ra612) mutation (G1442R) alters the temporal course of the P/Q-type current. The cloning strategy used to generate the mutant a1A construct involved deleting a portion of the polylinker from the 5' end of the pc3RBAl cDNA clone to yield moc1A. To ensure 167 Figure 21. The G1442R mutation affects the temporal course of the P/Q-type current and modifies the voltage-dependence of non-inactivating current. A and B) Representative whole cell traces of Ba 2 + currents through oc1A (A) and oc1A.612 (B) channels. Currents were evoked by stepping the membrane potential from -120 mV to voltages ranging from -60 mV to +25 mV in 5 mV increments. C and D) The extent of inactivation (r) for oc1A (C) and oc1A.612 (D) was plotted as a function of membrane potential. Data represents the mean \u00C2\u00B1 SE of the number of cells indicated in the figure. Note that the U-shaped voltage-dependence of r observed for ocIAis not present for ot]A_612, for which r decreases monotonically with voltage. 168 A B that this modification did not affect the expression of the construct, initial experiments comparing the electrophysiological properties of ma 1 A and pc3RBAlwere performed. Cells transfected with ma ] A expressed inward currents that did not differ significantly from currents detected in pc3RBAl-transfected cells (data not shown). Therefore, we concluded that the 22-bp deletion in the 5' polylinker of the pc3RBAl construct had no effect on expression or properties of the channel. Expression of ct1A_612 and wild-type a 1 A cDNAs in HEK tsa201 cells resulted in the expression VGCCs with markedly different electrophysiological properties. An obvious difference lies in the current waveforms (Figure 21 A and B). The wild-type and mutant channels differ markedly with respect to inactivation kinetics. Currents recorded from cells expressing a 1 A channels inactivated slowly during a test pulse to 0 mV (xinact = -200 ms). In contrast, a ] A_6 1 2 currents decayed rapidly, with a bi-exponential time course (xx = 14, x2 = 50). At the end of a 200 ms test pulse, wild-type and mutant currents had decayed by 42.92 \u00C2\u00B1 4.43 and 94.22 \u00C2\u00B1 1.16 %, respectively. These results are summarized in Table 10. The voltage-dependence of inactivation was also determined for the wild-type and mutant channels. The fraction of current resistant to inactivation (r) was calculated by dividing the amount of current remaining at the end of a test pulse (L ,^) by the maximum current obtained at the test potential (L,eak). Values of r were then plotted as a function of voltage (Figure 21 C and D). The voltage-dependence of inactivation of the wild-type current is U-shaped; the amount of current remaining at the end of the pulse reached a minimum value at moderate depolarizations (e.g. -5 to +10 mV) and increased again with stronger depolarizations. In contrast, over the potentials tested, the degree of inactivation of oc1A.6]2 decreased monotonically with depolarization, and reached a plateau at approximately 20% of the maximum current. 170 Table 10. Summary of the properties of oc]A and a a, Subunit YsO[ACT] (mV) ^ACT (mV) S^OIINACTJ (mV) ^INACT (mV) % inactivation I^NACT (ms) aiA -11.3 + 0.3 (n=5) 4.5 \u00C2\u00B1 0.2 (n=5) -52.5 + 0.2 (n=4) 5.6 + 0.1 (n=4) 42.92 \u00C2\u00B1 4.43 174.7 \u00C2\u00B1 25.7 aiA-612 -4.2 \u00C2\u00B1 0.2 (n=13) 4.2 \u00C2\u00B1 0.1 (n=13) -73.6 \u00C2\u00B1 0.4 (n = 6) 7.6 \u00C2\u00B1 0.3 (n = 6) 94.22 \u00C2\u00B1 1.16 9.16 \u00C2\u00B10.77 60.71 \u00C2\u00B1 7.85 171 Figure 22. The G1442R mutation alters the current-voltage relationship and steady-state inactivation properties of the channel. A) The oc1A.612 mutation produces a positive shift in the current-voltage (I-V) curve. The mean Ba 2 + current recorded from HEK tsa201 cells transfected with a 1 A (circles; n= 5) or a i A - 6 i 2 (squares; n= 13) Ca 2 + channel subunits. Data were normalized to the peak current observed in each cell and represent the mean \u00C2\u00B1 SE. B) The cx1A_612 mutation shifts the steady-state inactivation to more negative potentials. Membrane potential was stepped to the voltage that produces the maximal current in each condition from -120 mV after 15 sec. at holding potentials ranging from -102 mV to +20 mV. Normalized peak currents elicited by the test pulse were plotted as a function of the holding potential (a1A, circles; oc1A.612, squares). 172 173 G1442R produces a positive shift in the current-voltage relationship. Further comparisons of a 1 A. 6 1 2 and wild-type oc1A currents indicated differences in a number of other voltage-dependent properties. First, the current-voltage relationship for a i A - 6 i 2 i s shifted approximately 10 mV more positive than that of the wild-type channel (Figure 22A). In 5 mM Ba2+, currents through the wild-type oc1A channels first activated at approximately -30 mV and peaked at approximately -10 mV. In contrast, a ] A . 6 1 2 channels activated and peaked upon depolarizations to -20 and 0 mV, respectively. No significant differences were detected in the voltage dependence of activation (charge movement for: a ] A = 5.7 \u00C2\u00B1 1.2, n = 4; a 1 A . 6 1 2 = 5.9 \u00C2\u00B1 1.7, n = 13) and the extrapolated reversal potentials for each channel were similar (+30 mV), although the permeability properties of the mutant have not yet been examined. The shift to more positive potentials makes this analysis difficult because at this range of potentials, the contribution of outward cesium current is increased. G1442R shifts the steady-state inactivation to more negative potentials. The sensitivity of the channel to holding potential (SSinact) is also altered by the ra672 mutation (Figure 22B). Steady-state inactivation curves derived for wild-type and mutant channels demonstrate significant differences in the potential at which half of the current was inactivated (V5 0 [ i n a c t ]). Compared to a 1 A, the voltage-dependence of inactivation of oc1A_612 was shifted by approximately 20 mV to more hyperpolarized potentials (V 5 0 [ j n a c t ] for: a ] A = -52.47 \u00C2\u00B1 0.18 mV, n = 4; a 1 A . 6 1 2 = -73.64 \u00C2\u00B1 0.37 mV, n = 6). 174 Figure 23. cx I A 6 1 2 Ca 2 + currents display similar properties. A) Representative traces of whole cell Ba 2 + (top) and Ca 2 + (bottom) currents recorded from HEK tsa201 cells transfected with ct]A.612 subunit cDNA. Currents were evoked by stepping from a holding potential of -120 mV to membrane potentials ranging from -60 to +25 mV in increments of 5 mV. Cells were held at each potential for 150 ms. Note the accelerated decay of the current observed in Ba 2 + is also observed with Ca 2 + as the charge carrier. B) I-V curve of wild-type (open circles) and mutant (open squares) using Ca 2 + as the charge carrier. C) Steady-state inactivation curves for wild-type (open circles) and mutant (open squares) using Ca 2 + as the charge carrier. 175 176 Figure 24. The fraction of non-inactivating current (r) is independent of Ca 2 + concentration. A and B) Representative current traces for a 1 A (A) and cc1A.612 (B) using Ca 2 + (red trace) or Ba 2 + (blue trace) in the external solution, or 1 mM intracellular EGTA in the recording pipette (green trace). Currents were recorded during 150-ms long pulses to the voltages that evoked maximal currents in each ionic condition, from a holding potential of -120 mV. Traces were scaled to the same amplitude to compare the extent of inactivation during the test pulse. C) The fraction of non-inactivating current (r) was calculated by dividing the test voltage step (Iped) by the peak current (Ipeak) and displayed as a bar graph for the different ionic conditions tested (a ] A, hatched bars; oc1A.612, cross-hatched bars). 1 7 7 178 The effects of the G1442R mutation on channel properties are independent of the charge carrier. To assess the effect of different permeant ions on the properties of the mutant channel, the experiments described above were carried out using 5 mM Ca 2 + as the charge carrier (Figure 23A). Apart from an approximately 10 mV depolarizing shift in the I-V relationships and steady-state inactivation curves of both the mutant and wild-type channels (Figure 23B and C), the results of these experiments were similar to those discussed above. As was the case with Ba2+, currents recorded from cells transfected with cc1A.6!2 inactivated more rapidly than those recorded from cells expressing the wild-type channel. Again, the U-shaped voltage-dependence of inactivation observed for a 1 A was absent for a i A - 6 i 2 currents (data not shown). Figure 24 shows representative traces of a I A (A) or a 1 A . 6 1 2 (B) currents recorded in Ba 2 + and Ca 2 +. The third trace was also recorded in Ba2 +, except the effective intracellular divalent concentration was raised by lowering the concentration of the C a l -endaring agent EGTA in the pipette from 11 mM to 1 mM (see Methods and Materials). The fraction of residual current at the end of a 150-ms pulse in each ionic condition was displayed in a bar graph (C). Note that the fraction of non-inactivating current is not affected by altering the Ca 2 + concentration. The unc-2(ra611) mutation (G1254R) does not appear to affect the electrophysiological properties of exogenously expressed oc1B channels. The second missense mutation, identified in the unc-2(ra611) mutant, consisted of the substitution of an arginine for glycine 1254. To analyze the effect of this mutation, the corresponding amino acid in the rat brain a1B_n N-type channel was altered to an arginine. The two a, subunits were expressed in HEK tsa201 cells and their electrophysiological properties were examined. Figure 25A shows a family of current traces from cells 179 Figure 25. The G1254R mutation does not alter the macroscopic a 1 B . \u00E2\u0080\u009E current. A) Whole cell Ba 2 + currents recorded from HEK tsa201 cells transfected with wild-type (left) or mutant (right) a 1 B subunit cDNA. B and C) Neither the current-voltage relationships (B), nor the steady-state inactivation curves (C) of oc1B (triangles) and ocIB (stars) are significantly different. 180 181 expressing the wild-type and a1B_6I1 channels. The waveforms of both currents were very similar and both currents activated and inactivated with similar kinetics. To determine whether the mutation affected the voltage dependence of activation, the current-voltage relationships was examined for the two channels (Figure 25B). Cells transfected with a 1 B or a 1 B. 6 1 1 cDNA were stepped from holding potentials of -120 mV to potentials ranging from -30 to +65 mV. Both channels first activated at approximately -20 mV and peak current was attained at approximately 20 mV. Half-maximal activation (V 5 0 [ A C T ]) was calculated by fitting the data with the Boltzman equation (see Methods and Materials). Currents through both the wild-type and mutant channels reached 50% of the maximal current at 9.35 and 9.9 mV, respectively. The oc1B N-type VGCC is highly sensitive to holding potential. For example, at resting membrane potentials positive to -70 mV, approximately 50% of oc1B channels are in an inactivated state, and therefore unavailable for opening (Williams et al, 1992a; Dubel et al, 1994). To determine if the G1254R mutation affected this property, the sensitivity to holding potential of the mutant channel was compared with that of the wild-type channel. As shown in figure 25C, there was a small, non-significant shift of +4 mV in the SS inac t curve for the mutant with respect to the wild-type curve. In summary, the ra612 allele of unc-2 contains a substitution of an arginine for a glycine in the proximal portion of the carboxyl tail. The corresponding alteration introduced into the class A ax subunit from rat brain produces several striking changes in the biophysical properties of the oc1A current when expressed in a mammalian cell line. These include a markedly increased rate of inactivation of the whole cell current, as well as altered voltage dependent properties bf activation and inactivation. In contrast, the ra611 mutation, which consists of a glycine-to-arginine substitution in the IVS4 transmembrane segment, produced no significant detectable changes in the whole cell oc1B current recorded from HEK tsa201 cells. 182 Chapter 6. Discussion Molecular cloning of a VGCC a } subunit in the nematode C. elegans. The goal of this project was to develop a system in which VGCCs could be studied in an animal amenable to genetic manipulation. The ease with which C. elegans can be manipulated both genetically and molecularly makes it an attractive candidate organism for such a system. When I began this study, VGCC subunit proteins had not yet been identified in C. elegans, although there was some evidence for their existence (Willett et al, 1991; Lobel and Horvitz, 1993). I initially used a molecular biological approach to identify a VGCC a, subunit in C. elegans. Sequence comparisons of VGCC a; subunits from vertebrates revealed regions of high levels of amino acid identity and allowed degenerate primers to be used to amplify homologous sequences from C. elegans RNA. Southern blots of genomic DNA hybridized with the PCR product Ce2 showed a simple pattern of hybridizing fragments. Furthermore, Ce2 hybridized to three overlapping clones on a YAC grid, localizing the gene to the left arm of the X chromosome and suggested that there was a single VGCC a, subunit gene related to the mammalian VGCCs. However, at roughly the same time, another group amplified a C. elegans VGCC a, subunit PCR product which hybridized to two loci on the YAC grid, placing the second putative VGCC gene on LGIV near the egl-19 locus (Lobel et al, 1993). Interestingly, 183 neither fragment hybridized to loci identified by the other. A comparison of the DNA sequences in the Ce2 region of unc-2 and egl-19 reveals that the two genes share less than 50% overall identity, and there is even less over the approximately 900-bp egl-19 fragment used to screen the YAC grid by Lobel et al. (1993). Since the completion of the genome sequence by the C. elegans Sequencing Consortium, it has become clear that there are at least three genes encoding VGCC a, subunits in C. elegans (Table 3). Sequence comparisons suggest that these channels may be viewed as representatives of the three major channel types described in vertebrate systems: the HVA DHP-insensitive N-, P/Q- and R- types, the DHP-sensitive L-types, and the LVA T-types (Figure 11; Table 5). According to this scheme, UNC-2 is predicted to be a high threshold DHP-insensitive channel, EGL-19 an L-type channel, and C54D2.5 a T-type channel. These classifications for UNC-2 and EGL-19 are supported by more detailed sequence analyses and genetic studies (see below). Currently, the only evidence supporting the hypothesis that C54D2.5 encodes a T-type channel comes from the fact that this sequence was used to clone vertebrate channels possessing the properties expected of T-type channels (Perez-Reyes etai, 1998; McRory etai, 1999; Santi etai, 1999). Genomic organization of the VGCC a t subunit encoded by the unc-2 gene. The Ce2 channel that I identified was subsequently shown to correspond to the unc-2 locus (Schafer and Kenyon, 1995). The unc-2 gene spans approximately 25 kb and consists of 28 exons that potentially encode a 1992-amino acid polypeptide. The sequence and predicted structure of this polypeptide are very similar to those of HVA VGCCs (see below). There are several noteworthy features of the genomic organization of the unc-2 gene and its protein product. The first two introns in unc-2 are quite large, spanning 5434- and 4981-bp, respectively. Although introns in C. elegans are most frequently smaller than 60-184 bp in length, many genes contain large introns near the 5' end. In some cases, these introns have been shown to contain regulatory elements such as alternative promotors or transcriptional enhancers (reviewed in Blumenthal and Steward, 1997). It is also noteworthy that the VGCC homologs encoded by EGL-19 (C48A7.1) and the C54D2.5 genes contain large introns near their respective 5' ends. There is no obvious relationship between the intron-exon structure of the gene and domains or other structural features of the unc-2 VGCC. For example, domain I is encoded by five exons, and each of these exons encodes a different number of transmembrane segments. By comparison, domain IV is encoded by only two exons. Furthermore, some exons encode portions of adjacent domains. For example, exon 17 encodes the last two transmembrane segments from domain III and the first two segments of domain IV. The voltage-gated Ca 2 + and Na+ channels, which consist of four homologous domains, are thought to have evolved from the single-domain voltage-gated K + channels through duplication of the transmembrane domain. While this may be the case, the unc-2 genomic structure has diverged substantially from that of the primordial voltage-gated ion channel. unc-2 likely encodes an HVA, DHP-insensitive V G C C . The longest open reading frame of the unc-2 gene encodes a 1992-amino acid polypeptide with a predicted molecular weight of 227 kDa. Comparison of the UNC-2 sequence with those of vertebrate VGCCs indicates that it is a nematode homologue of an HVA VGCC. At 1992 amino acids, UNC-2 is smaller than the vertebrate neuronal, cardiac, and smooth muscle VGCCs, which range in size from 2143 to 2424 amino acids, and slightly larger than the 1873-amino acid rabbit skeletal muscle VGCC (Stea et al, 1995a). The predicted UNC-2 protein consists of four homologous transmembrane domains interspersed with predicted cytoplasmic and extracellular regions of varying lengths. In addition to the high 185 level of sequence conservation in the four membrane spanning domains, regions such as the pore-forming loops, the positive charges in the voltage-sensing S4 segment, the P subunit binding motif in the I-II linker, and the EF hand-like motif near the carboxyl terminus are also conserved with mammalian HVA VGCCs. At the amino acid level, UNC-2 is more similar to the non-L (ocIB, a I A , and aIE) N-, P/Q-, and R-type channels than to the L-type (a l c, a m , and oc!F) and T-type channels (a,G, oc1H, and ocn). Overall, UNC-2 shares more sequence similarity with the non-DHP-sensitive channels from rat brain than with the two other VGCCs from C. elegans (Table 5). An insensitivity to DHPs and phenylalkylamines is a defining characteristic of non-L-type channels. Binding experiments (Nakayama etal., 1991; Striessnig etal., 1990) implicate the region encompassing residues in the IIISS2-IIIS6 and IVSS2-IVS6 of L-type channels in DHPs binding. In the homologous region in the UNC-2 protein (158 amino acids), there are 43 non-conserved residues. The phenylalkylamine binding site has been localized to a 44-amino acid region spanning IVS6 and extending into the carboxyl tail (Striessnig et al., 1990). All VGCCs cloned to date are highly similar in this region; only five positions differ between the DHP-sensitive and insensitive channels. Four of these five residues are critical for conferring DHP- and phenylalkylamine sensitivity (Schuster et al, 1996). The residues in the homologous positions in UNC-2 are identical to those found in the non-DHP sensitive channels (Figure 13B). The absence of a strong consensus in the putative DHP-binding region of UNC-2 supports the prediction that this channel is a member of the DHP-insensitive family of VGCCs. A careful examination of the class A, B, and E protein sequences reveals that there are regions in which residues are conserved between only two of the three channel classes, and others in which the three channels differ from one another altogether. While UNC-2 shares the most sequence identity with the class B channel, in locations where these two channels differ, UNC-2 frequently contains the identical or conserved residue found in either the class A or E channels. One dissimilarity between UNC-2 and vertebrate DHP-186 insensitive channels lies in the cytoplasmic linker connecting Domains II and lU. In vertebrates, the A, B, and E a, subunits have long cytoplasmic linkers (479 - 539 amino acids) connecting these domains. The corresponding segment in UNC-2 is only 103 amino acids, making it more similar in length to the DHP-sensitive aj (115 -150 amino acids). Finally, the UNC-2 channel has a different pattern of charge placement in the S4 membrane-spanning segment than all the vertebrate VGCCs. The UNC-2 S4 segments in domains I and III are almost identical to their vertebrate counterparts. However, IIS4 and IVS4 have one less charge than the equivalent segments in the mammalian channels. In both instances, the carboxyl-most charge is replaced with a serine residue. UNC-2 does not appear to be exactly analogous to a specific channel type. In many respects, the UNC-2 VGCC appears to be most closely related to the vertebrate DHP-insensitive VGCCs, although it does contain features characteristic of DHP-sensitive channels. It is possible that UNC-2 represents a channel with properties intermediate to these different types. Functional expression of UNC-2 in a surrogate system such as Xenopus oocytes or mammalian tissue culture cells will be necessary to characterize its electrophysiological and pharmacological properties and assign it to a particular pharmacological class of VGCC. The UNC-2 V G C C a, subunit appears to be expressed as a single isoform. A single transcript of approximately 7.5 kb was detected on Northern blots of staged RNA, suggesting that UNC-2 is expressed as a single isoform throughout C. elegans development. This is in contrast to the situation seen in mammals and Drosophila, in which several transcripts of significantly different sizes are detected on Northern blots (Snutch et al, 1990; Snutch et al, 1991; Fujita et al, 1993; Zheng et al, 1995). It is possible that alternative splicing of the primary transcript occurs on a smaller scale, resulting in small insertions or deletions such as those described for both the oc1A and a 1 B 1 8 7 genes (Bourinet et al, 1999; Stea et al, 1999) and are not resolved by Northern blotting. However, sequence analysis of multiple cDNA clones and PCR products have not indicated the presence of any splice variants to date. Isolation and characterization of novel mutations in the unc-2 locus. The identification of unc-2 as a VGCC made it possible to design a mutant screen to identify novel alleles of the gene, unc-2 mutants display mild behavioral abnormalities, including slow, kinked movement, difficulty in backing, and egg-laying defects (egg-laying constitutive). I isolated eleven EMS-induced alleles using a precomplementation screen. These mutants were all uncoordinated, but to varying degrees of severity. A more detailed phenotypic analysis was undertaken with three strains in which the molecular lesion was identified. In these cases, I tested the mutants' responses to drugs such as aldicarb and nicotine, and made double mutants with other mutations in other genes that may be physically associated with UNC-2 or involved in similar physiological pathways. The success of the unc-2 mutant screen illustrates the potential of this model system for VGCC analyses. The detection of eleven alleles amongst only 20,000 animals suggests that this is an efficient way to generate mutations in VGCCs. Since we were interested in examining the effects of small, localized alterations to the channel protein, I chose to mutagenize the animals with EMS which produces primarily point mutations by causing G/C to A/T transitions. However, a different array of mutations may be obtained by using other agents that affect C. elegans (Johnsen and Baillie, 1997). For example, ENU predominantly causes AT to GC transitions which would result in a different spectrum of changes than EMS. In particular, ENU is predicted to make more missense mutations, and this could be especially useful in analyzing structure-function relationships. In addition, different screening strategies can be utilized to isolate alleles with specific attributes. For example, screens for alleles which confer different sensitivities to pharmacological agents 188 known to act on VGCCs may provide a powerful tool for determining the site and mode of action of these drugs. I identified the molecular lesions in four of the mutants derived from the precomplementation screen (ra605, ra610, ra611, and ra612) and three mutants obtained from the Rand Laboratory (md328, mdl064, and mdll86). The lesions in ra605, ra610, ra611, and ra612 are all G/C to A/T transitions as expected for EMS-induced mutations. The md328 allele was a spontaneous mutation derived from the RM25 strain (J. B. Rand, personal communication). mdl064, and mdll86, also spontaneous mutations, are derived from TR638, a mut-3 strain with a high frequency of germ-line Tel transposition (Miller et al, 1996). Three of the alleles, ra605, ra610, and md328, are likely to be null. ra605 and ra610 each contain nonsense mutations that result in a truncation of the protein in the fourth transmembrane domain. The md328 allele contains a complex rearrangement that destroys the 3' splice acceptor site in intron 12. At the very least, this mutation would result in the deletion of half of the IIS 6 transmembrane segment and result in a non-functional protein. Tel elements were located in exon 3 of mdll86 and in exon 18 of mdl064. Both alleles were screened for spontaneous revertants, which resulted in the recovery of an eighth allele, unc-2(ra614). This allele arose from the RM1064 strain and contains a missense mutation that results in the conversion of tyrosine 1254 to a cysteine. The ra611 and ra612 alleles isolated in the EMS pre-complementation screen both contain missense mutations. The ra612 mutation results in the alteration of glycine (G) 1442 to an arginine (R), while in ra611, glycine 1254 is altered to arginine. Glycine 1442 is located in the carboxyl terminal region of the protein, nine amino acids downstream of the putative EF-hand motif. In all VGCCs cloned to date, the homologous residue is always a glycine. The same is true for the glycine mutated in ra611, except that at this position in the rabbit ccls channel, a conserved substitution of an alanine is found. The conservation of these amino acids, combined with the aberrant phenotypes manifested upon mutation, suggests that these two residues are essential to normal channel function. To 189 assess the consequences of these mutations on channel properties, I introduced the corresponding changes into the class A and B a, subunits cloned from rat brain (see below). UNC-2 is involved in acetylcholine and GABA neurotransmission. Animals with null or reduction-of-function mutations in the unc-2 gene display mild behavioral abnormalities, including slow, kinked movement, difficulty in backing, and egg-laying defects (egg-laying constitutive). This phenotype is similar to that of mutants known to be defective in neurotransmission (Rand and Nonet, 1997) and suggests that unc-2 is also involved in this process. Furthermore, unc-2 animals are aldicarb resistant (Miller et al, 1996; E. A. Mathews, this study), implicating UNC-2 specifically in cholinergic neurotransmission. The [essentially] wild-type response of unc-2 animals to levamisole (Miller et al, 1996), indicates that the aldicarb resistance is not due to an abnormal response to ACh by the post-synaptic cell. While there is evidence (Schafer and Kenyon, 1995) that unc-2 is expressed in the body wall muscles, null alleles of unc-2 do not result in a Pat (paralyzed arrest at embryonic two-fold stage) phenotype, which is typical of essential muscle genes (Williams and Waterston, 1994; reviewed in Moerman and Fire, 1997). By comparison, null mutations in egl-19 result in a Pat phenotype, suggesting that EGL-19 is the major VGCC a, subunit in the body wall muscles (Lee et al, 1997). Taken together, these observations indicate that unc-2 plays a minor role, if any, in initiating body wall muscle contraction and instead suggest that UNC-2 functions presynaptically to regulate the release of ACh. In addition to aberrant locomotory and egg-laying behaviors, unc-2 mutants are defective in the expulsion step (EMC) of the defecation cycle (Miller et al, 1996; E. A. Mathews, this study). The AVL and DVB neurons that stimulate the contraction of the enteric muscles are GABAergic. GABA is also released from a set of motor neurons to produce contralateral inhibition of the body wall muscles (Mclntire etai, 1993[a,b]). 190 Furthermore, aspects of the mutant phenotypes of unc-46 and unc-47, two genes required for normal GABA release, are similar to those of unc-2. For example, these three mutant phenotypes share slow forward movement, and very poor backwards movement. Thus, the unc-2 gene product appears to play a role in the release of GABA as well as ACh, and may have a generalized role in neurotransmission in C. elegans. Behavioral and pharmacological studies suggest that UNC-2 functions in the presynaptic neurons of C. elegans in a manner analogous to the N- and P/Q-type channels in vertebrate nervous systems. Null mutations in cha-1 and unc-17, which encode choline acetyltransferase and a synaptic vesicle ACh transporter, respectively, completely abrogate ACh release. These mutants are inviable; they hatch, but are paralyzed, rendering them barely able to move or feed, and they arrest development at the L l stage (Nonet et al, 1993; Rand and Nonet, 1997). The unc-2 null phenotype (ra605, ra610, md328) is significantly milder than that of cha-1 and unc-17, suggesting that even in the absence of unc-2 there is release of ACh from motor neurons. This conclusion is further supported by the observation that unc-2 animals became hypercontracted after prolonged exposure to aldicarb, suggesting that some ACh release does occur. Thus, if unc-2 is involved in ACh release from the motor neurons, it cannot be the sole mediator of ACh release at the NMJ. There are several other VGCCs in C. elegans that could potentially mediate neurotransmission. One of these, the putative L-type channel encoded by the egl-19 gene, is thought to act predominantly in muscle; however, it is expressed in at least a subset of neurons (Lee et al, 1997) and it is possible that it plays a role in neurotransmission. Another possibility is that the putative T-type channel encoded by C54D2.5 functions in this process. However, no mutations have been identified in this gene to date nor have any mutations affecting synaptic transmission been mapped to this region of the genome. Alternatively, the novel VGCC-like proteins encoded by C27F2 and Cl 1D2 may be involved in cholinergic transmission. However, null mutations in these genes result in only mild locomotory and behavioral defects (G. P. Mullen, personal communication.). It 191 is possible that the various channel proteins have partially overlapping/redundant roles in cholinergic transmission. This hypothesis could be tested by constructing various combinations of double and triple mutants with each other and with unc-2. Given that ACh release is essential for viability in C. elegans (Rand and Nonet, 1997), it is reasonable to speculate that there is redundancy built into this system. A further possibility is that the locomotory phenotype exhibited by unc-2 mutants arises through abnormal neurotransmitter release at the level of the interneurons. If this were the case, loss of unc-2 function might be expected to uncouple the excitatory and inhibitory impulses onto opposing body wall muscles that are necessary for coordinated movement without affecting release from the motor neurons. In this case, another mechanism would be responsible for ACh release from the motor neurons, while unc-2 could be solely responsible for release from the interneurons. Without a clear picture of the expression pattern of unc-2, it is difficult to determine its precise role in C. elegans neurophysiology. Attempts at in situ staining using a polyclonal serum directed against the unique II-III loop of UNC-2 were unsuccessful. However, construction of a green fluorescent protein (GFP)::promotor fusion construct would be very useful in identifying the cells in which unc-2 functions. In addition, if loss of unc-2 prevents transmitter release from a subset of neurons, one might expect an accumulation of transmitter in the presynaptic cell. This accumulation could be detected with antibodies against the neurotransmitter, as has been noted with respect to GABA in unc-47 animals (Mclntire et al, 1993a). Testing for increased GABA immunoreactivity in unc-2 animals could provide evidence for a role in synaptic release. UNC-2 and UNC-36 may function in the same V G C C complex. The unc-36 gene encodes a polypeptide that is highly similar to the a2/8 subunits expressed in mammalian nervous systems (Lobel and Horvitz, 1993). Several observations suggest that UNC-2 and UNC-36 function as part of the same VGCC complex. Mutations in the 192 unc-36 gene result in movement and egg-laying defects that are indistinguishable from those exhibited by unc-2(null) mutants, unc-2 and unc-36 mutants also exhibit very similar responses to the AChE inhibitor aldicarb (Nguyen et al, 1995) and to the presence of exogenous neurotransmitters, including dopamine and serotonin. In addition, unc-36 and unc-2 mutants exhibit similar defects in adaptation to these compounds (Schafer and Kenyon, 1995; Schafer etal, 1996). Mutations in these two genes also exhibit similar genetic interactions; unc-36; egl-19 and unc-2; egl-19 double mutants are both essentially paralyzed and are indistinguishable in this respect (Schafer et al, 1996; E. A. Mathews, this study). Finally, unc-36; unc-2 double mutants are indistinguishable from either single (null) mutant, suggesting that these genes function in the same pathways (Schafer et al, 1996; E. A. Mathews, this study). These observations also suggest that UNC-36 activity is required for the functional expression of the UNC-2 VGCC complex. As noted above, unc-2 and unc-36 mutations result in very similar phenotypes and unc-36; unc-2 double mutants are indistinguishable from either single mutant. Since the absence of unc-36 (a2/8 subunit) function has the same effect as the absence of unc-2 (a, subunit) function, the a2/8 subunit must be essential for the activity of the complex. This conclusion is consistent with observations made on VGCCs expressed in mammalian tissue culture cells, where it was found that co-expression of a cloned a2/8 subunit is necessary for the expression of detectable Ca 2 + currents in most exogenous expression systems. As noted previously, the egl-19 gene encodes a VGCC a, subunit most closely related to the DHP-sensitive HVA subunits (Lee et al, 1997). Potentially, the UNC-36 and EGL-19 proteins could also form a VGCC complex in vivo. Although the movement and egg-laying defects exhibited by unc-36 and egl-19 mutants are quite distinct, mutations in both of these genes result in hypersensitivity to the L-type VGCC antagonist verapamil (Lobel et al, 1994). In addition, unc-36; egl-19 double mutants exhibit a paralyzed phenotype (Schafer et al, 1996; E. A. Mathews, this study). Although these observations 193 are suggestive of an in vivo interaction, a persuasive case can be made that these proteins chiefly function in different VGCC complexes and distinct biological processes. The loss of function phenotypes of unc-36 and egl-19 are quite distinct; unc-36 mutants are uncoordinated and possess characteristics typical of mutants with impaired synaptic function, whereas egl-19 mutants are Pat (paralyzed arrest at embryonic two-fold stage), a phenotype typical of mutations affecting essential muscle components (reviewed in Moerman and Fire, 1997). In addition, the unc-36; egl-19 double mutant phenotype is indistinguishable from that of unc-2; egl-19 double mutants and, therefore, probably does not reflect an in vivo association. A possible explanation for these results is that UNC-2 and UNC-36 interact in vivo and participate principally in neurotransmitter release. In contrast, EGL-19 functions chiefly in the muscles where it plays an essential role in initiating muscle contraction. It should be noted that there is at least one additional a2/8 subunit, encoded by T24F1.6, which could conceivably interact with EGL-19 in the muscles. The paralyzed phenotype of unc-2; egl-19 and unc-36; egl-19 double mutants could reflect the net effect of reduced neurotransmitter release and reduced Ca 2 + influx in response to neural signaling. According to this model, the sensitivity of egl-19 mutants to verapamil could reflect the reduced abundance or function of VGCCs in the muscles. In turn, the sensitivity of unc-36 mutants to verapamil may reflect the reduced neurotransmitter release observed in these animals, which would reduce the degree of depolarization by the post-synaptic muscle cells. The net effect of this reduction in cell depolarization is a reduction in Ca 2 + influx, possibly by reducing the degree of channel activation. In this situation, the reduced activity of the VGCCs in the muscles could result in an apparent increase in sensitivity to inhibition by verapamil. Therefore, in both unc-36 and egl-19 mutants, the heightened sensitivity to verapamil could reflect the reduced activity of the muscle L-type VGCC. A prediction of this hypothesis is that unc-2 and other mutants with impaired synaptic function should also be hypersensitive to verapamil because they also result in decreased muscle activation via 194 reduced neural signaling. It is likely, however, that this model is an oversimplification; egl-19 is expressed in at least a subset of neurons (Lee et al, 1997) and mosaic analysis suggests that unc-2 has a minor role in the body wall muscles (Schafer and Kenyon, 1995). In addition, unc-2 and unc-36 mutants have at least two subtle phenotypic differences: i) unc-2 mutants exhibit Q cell migration defects, which are not observed in unc-36 mutants (Tarn et al, submitted), and ii) unc-2 males mate quite efficiently, whereas unc-36 males are incapable of mating. These differences may well reflect tissue-specific interactions between UNC-2 or UNC-36 and other VGCC subunits in C. elegans. Analysis of unc-2 mutations in a heterologous expression system. Of the eight unc-2 alleles analyzed in this study, ra611 and ra612, contain missense mutations that would be expected to affect the function of the channel by altering its electrophysiological properties or its sensitivity to modulation. Both of these alleles result from an G/C to A/T transition that changes a glycine to an arginine. In each case, the altered glycine is conserved in virtually all VGCCs cloned to date. The conservation of these amino acids throughout evolution suggests that these residues are functionally significant. To understand how these alterations impact channel activity, I introduced them into the rat brain oc]A (ra612) and a 1 B (ra611) subunit clones. I chose to study these mutations in the class A and B channels as they are the closest vertebrate homologs to the C. elegans UNC-2 channel. The ra612 mutation alters a number of P/Q-type channel properties The mutation in the ra612 allele results in the substitution of glycine 1442 with an arginine. This mutation is located near the putative EF-hand motif in the channel tail, 55 residues carboxyl to segment IVS6. The corresponding mutation was introduced into the a 1 A P/Q-type subunit from rat brain and transiently transfected into FfEK tsa201 cells. Currents recorded from cells expressing mutant channels inactivated considerably more rapidly than 195 wild-type currents. In addition, the current-voltage relationship of the mutant was shifted approximately 10 mV more positive. Thus, a stronger depolarization is required to open the mutant channels which, once open, inactivate more rapidly than wild-type, thereby terminating the Ca 2 + influx sooner. Furthermore, the mutation induces an approximately -20 mV shift in the steady-state inactivation of the channel. For example, 50% of wild-type channels in a cell with a resting membrane potential of approximately -52 mV are capable of opening, whereas over 90% of the mutant channels are inactivated at the same potential. This dramatically reduces the number of channels available for opening from a given membrane potential and would further attenuate Ca 2 + influx during a depolarization. It is necessary to be cautious when inferring the biological implications of the ra612 mutation from results obtained from experiments performed in surrogate expression systems as we neither know the electrophysiological properties of the UNC-2 channel nor the efficiency of the cellular mechanisms in the C. elegans neuron responsible for Ca 2 + buffering and sequestration. The unc-2(ra612) animals were significantly less defective in thrashing and defecation than were putative unc-2 null mutants. Thus, these electrophysiological results are consistent with the phenotypic observations that suggest that the ra612 mutation does not result in the complete elimination of channel function. Ca 2 + influx into cells modulates membrane excitability, and intracellular Ca 2 + acts as a second messenger, activating Ca2+-dependent enzymes and triggering exocytotic release of neurotransmitters (reviewed in Miller, 1987). Thus, the decrease in neuronal activity in unc-2(ra612) animals could be attributed to diminished Ca 2 + influx through the mutant channels. For example, the increased rate of inactivation would reduce the duration of Ca 2 + influx resulting in less transmitter release. This decrease would attenuate muscle contraction and could result in the uncoordinated phenotype. 196 The fast inactivation seems to be a property of the a 1 A . 6 1 2 subunit. We have currently begun to explore the mechanisms underlying the changes in inactivation properties of the a 1 A channel. A number of studies have shown that the (3 subunits differentially modulate the inactivation kinetics and voltage-dependent properties of the a, subunits (Stea et al, 1994; Stea et al, 1995b). Preliminary results indicate that the a,A and a i A - 6 i 2 constructs are similarly affected by the (3,b and (32 subunits. For both a, subunits, co-expression of the P2 subunit slowed inactivation as compared to P l b. In addition, the p2 subunit shifted the voltage-dependence of inactivation to more positive potentials. While the properties of the oc,A_612 alone need to be assessed, and it remains to be seen how the P3 and P4 subunits affect the properties of the oc1A.612, the mutant appears to be modulated by (3 subunits in the same manner as the wild-type channel. Because the increase in inactivation and shift in voltage-dependence of the oc1A.612 channel was seen with both the p l b and (32a subunits, the differences between the mutant and wild-type channels must be properties inherent to the a, subunit, and not dependent on the expression system or auxiliary subunits. However, it is possible that the mutation alters other modulatory sites, either by destroying them, or by producing conformational changes that prevent the binding of interacting proteins. An interesting aspect of P subunit modulation is derived from a pair of studies which demonstrate that [at least in vitro] the p4 and, to a lesser extent the (32, subunit interact specifically with the carboxyl-terminal region of the oc1A (Walker et al, 1998; Walker et al, 1999). It is interesting to note that, while coexpression of (32 produces the expected reduction in the mutant channel's rate of inactivation, the effect of (32 on the kinetics of the mutant channel is much weaker than on the wild-type. It is possible that the ra612 mutation weakens the interaction between the oc1A and (32a subunits. While the ra612 mutation is not located in the regions thought to bind the p subunit (Walker et al, 1999), the mutation may induce a localized conformational change in the channel which interferes with P subunit binding to secondary sites near the carboxyl tail. Because the P4 has been 197 shown to bind even more strongly to the cc1A than the p\>, it will be interesting to determine whether p4 modulates the properties of a 1 A. 6 1 2 in the expected manner. The increased rate of inactivation does not appear to be Ca2+-dependent. Two independently-functioning mechanisms have been described for inactivation of VGCCs (Hille, 1992). All VGCCs undergo voltage-dependent inactivation, which is primarily regulated by membrane potential, and is thought to affect the voltage-sensing mechanism directly. Voltage-dependent inactivation increases with the strength of the depolarization and peaks at potentials at which the channels are maximally activated. In this case, the rate of inactivation is independent of the species of ion carrying the current and the amount of current passing through the channel. In the second mechanism, Ca2+-dependent inactivation, the accumulation of intracellular Ca 2 + attenuates the current. Ca2+-dependent inactivation is characterized by several properties: i) the degree to which channels inactivate decreases as the depolarization approaches the reversal potential of Ca 2 +, reducing the magnitude of the Ca 2 + influx, thereby producing a U-shaped curve when the percent inactivation (or the x of inactivation) is plotted against voltage, ii) current decay is more rapid with Ca 2 + as the charge carrier, as opposed to other channel-permeable ions, such as Ba 2 + or S T 2 * , iii) increasing extracellular Ca 2 + concentration (and therefore the magnitude of the Ca 2 + current) further increases the rate of inactivation, and iv) inactivation is reduced by compounds such as EGTA that chelate intracellular Ca 2 + (Jones, 1999). An early model of Ca2+-dependent inactivation involved the Ca2+-activated phosphatase, calcineurin, which dephosphorylates the channel and reduces its activity (Chad and Eckert, 1986; reviewed in Hille, 1992). More recent work favors a model in which Ca 2 + binds directly to the channel (Yue et ai, 1990; Haack and Rosenberg, 1994; Imready and Yue, 1994; Neely et al, 1994). The general consensus is that the carboxyl terminal end of the channel contains the machinery responsible for Ca2+-dependent inactivation, although the mechanism remains unclear. Experiments on chimeras 198 constructed between the a i c and the a I E (a channel that does not possess Ca2+-dependent inactivation) (de Leon et al, 1995; Zhou et al, 1997) and on a ] C splice variants (Soldatov et al, 1997; Soldatov et al, 1998) demonstrate that a 216-residue region beginning with the EF-hand in the C-terminus of the a i c is necessary for Ca2+-dependent inactivation. Ziihlke and Reuter (1998) refined these results to identify three specific sequences contained within this region that are required for Ca2+-dependent inactivation. These three sequences include the EF-hand, two downstream sequences consisting of an asparagine-glutamate (NE) pair and an 8 residue stretch that was of great interest because of its similarity with the consensus motif (IQ motif) for calmodulin binding. Recent studies (Peterson et al, 1999; Ziihlke et al, 1999) have shown that mutant calmodulin eliminates Ca2+-dependent inactivation, suggesting a model in which calmodulin is constitutively bound to the channel [most likely at the IQ motif] and, once bound to Ca 2 +, induces a conformational change in the a, subunit that favors channel closure. Ca2+-dependent inactivation is most commonly associated with L-type VGCCs. However, it has also been implicated as a mode of inactivation of N- and P/Q-type channels (Cox andDunlap, 1994; Tareilus et al, 1994; Shirokov, 1999). Furthermore, calmodulin has been shown to bind in the carboxyl tail region of the cx1A, and this binding appears to be involved in Ca2+-dependent modulation of P/Q-type channel (A. Lee et al, 1999). Because there are trace amounts of Ca 2 + in the salts used to prepare the recording solutions, the inactivation observed with Ba 2 + currents likely results from this contamination. Some studies have also suggested that the mechanism responsible for Ca2+-dependent inactivation may also be responsive to Ba 2 + , although at a much lower sensitivity (Cox and Dunlap, 1994; Ferreira et al, 1997). Because the ra612 mutation is located in the vicinity of the regions implicated in Ca27calmodulin-dependent inactivation, we tested the possibility that the increased rate of inactivation of the a 1 A. 6 1 2 current results from the introduction or enhancement of a Ca2+-dependent component of inactivation. 199 If the inactivation of the oc1A.612 channel is due to activation of a Ca2+-dependent mechanism, then currents carried by Ca 2 + would be expected to inactivate faster and more completely than Ba 2 + currents. However, as the kinetics of current decay are similar regardless of Ca 2 + concentration (Table 10; Figure 24), it appears that the increased current inactivation is not due to a simple Ca2+-dependent mechanism. However, as noted above, inactivation could be mediated through a mechanism sensitive to all divalent cations. If this is the case, the rate of inactivation should be slowed when the permeant ion is monovalent. Preliminary results suggest that the rate of inactivation of Na+ currents recorded from cells expressing a 1 A . 6 1 2 currents is equal to those seen with Ba 2 + and Ca 2 + (data not shown). Finally, a plot of non-inactivating current as a function of voltage reveals that the U-shape curve observed for cc1A is not present with oc1A.612 (Figures 21C and 21D). This suggests that while there may be some degree of Ca 2 +- or current-dependent inactivation of the wild-type channel as suggested by Tareilus et al. (1994), Ca2+-dependent inactivation does not contribute to the decay of the oc1A_612 current. Future studies on the unc-2(ra612) mutation. In this study, I identified a mutation that changes the voltage dependent properties and increases the rate of inactivation of the P/Q-type channel. One of the difficulties in deciphering the exact nature of the changes induced by the mutation arises from the fact that very little is known about inactivation of VGCCs other than the L-type. Furthermore, unlike voltage-gated Na+ and K + channels, the mechanisms underlying voltage-dependent inactivation of VGCCs are poorly understood. The mechanism underlying the increased rate of inactivation does not appear to be a direct alteration of Ca 2 +- or current-sensitive properties of the channel. Potentially, the ra612 mutation might alter or occlude sites that interact with modulatory proteins that may or may not be Ca2+-activated. For example, a CamKinase II/PKA consensus sequence is located a few amino acids away from the mutation site. Phosphorylation of this site, which may serve to activate or facilitate the 200 P/Q-type current, might be inhibited by the mutation. Further studies using specific inhibitors of proteins known to modulate the P/Q-type channel such as PKA and calmodulin may help elucidate a possible mechanism. Further mutational studies in this region may also further our understanding of how the ra612 mutation alters the properties of the P/Q-type channel. In this study, I endeavored to identify second-site mutations in C. elegans that could \"suppress\" the phenotypic defects associated with the ra612 mutation. Although these studies were not successful, larger or more sensitive screens could be performed which may facilitate the identification of such mutations. Alternatively, specific alterations could be introduced into the P/Q-type channel by site-directed mutagenesis. There are a number of possible explanations for the change in properties associated with the ra612 mutation, and these could be tested directly using this approach. For example, the residue altered by this mutation is followed by two [positively-charged] lysine residues (GLGKKC\u00E2\u0080\u0094> GLRKKC). Potentially, the effect of the mutation could be due to the presence of an additional positive charge in this region of the protein. Alternatively, the substitution of an amino acid with a large R-group for one with a small side chain could interfere with proper protein folding. This could have direct effects on basic channel properties or protein interactions, or result in a conformational change that occlude sites of interaction with other proteins. The effects of such conformational changes could affect sites of interaction located considerable distances from the altered residue. The first hypothesis could be tested either by removing one of the adjacent charged residues to restore the net charge in the region, or by replacing the arginine with a large uncharged amino acid such as isoleucine. The replacement of arginine with a large neutral amino acid would also address the second hypothesis. 201 The ra611 mutation does not alter the basic electrophysiological properties of the o c 1 B N-type channel. Like the ra612 mutation, the ra611 sequence alteration is a glycine to arginine substitution. According to the predicted membrane topology, this glycine residue is thought to reside in the membrane as part of the S4 transmembrane segment (Tanabe et al, 1987; Stea et al, 1995a). Current models of voltage-gated ion channel activation assign to the S4 segment the role of \"voltage-sensor\"; this segment transduces changes in membrane potential into a conformational change in the protein which results in opening of the channel pore. The S4 segment has been shown to physically move through a transmembrane pathway formed by the rest of the channel protein (Yang and Home, 1995; Aggarwal and MacKinnon, 1996). This movement of charge as the S4 segment travels across the membrane produces a tiny, but detectable, current known as the gating current. The size of this gating current is determined by the number of charges that move in response to changes in membrane potential, and the number of charges that are translocated through the membrane determines the voltage dependence of activation of the channel (reviewed in Yellen, 1998). Researchers investigating the involvement of the S4 segment in activation have found that neutralizing many of the positively-charged amino acids produced positive shifts in the voltage-dependence of activation, reductions in the steepness of the activation curve, and smaller gating current (Stuhmer et al, 1989; Papazian et al, 1991; Aggarwal and MacKinnon, 1996; Seoh et al, 1996). These changes reflect a decrease in the sensitivity of the channel to changes in membrane potential. Since decreasing the net charge in the S4 region decreases voltage sensitivity, an increase in charge as is created by the G1254R mutation would be expected to increase the voltage sensitivity. However, the G1254R mutation had little, if any, impact on whole cell N-type current properties. The G1254R mutation was introduced into the rat brain a 1 B subunit protein and expressed in HEK tsa201 cells. Apart from an insignificant shift in the voltage-dependence of inactivation, no differences from wild-type were detected. Whole cell Ba 2 + currents 202 recorded from cells expressing mutant constructs were virtually identical to wild-type currents and the I-V relationships for the two channels are indistinguishable; both channels reached half-maximal activation at approximately 9.5 mV with a slope of 4.5 to 5 mV. Thus, it does not appear that the mutation alters the voltage sensitivity or other obvious electrophysiological properties of the channel. The slope of the activation curve is an indirect measurement of the gating charge that is accurate only for a simple system consisting of a single voltage sensor. In situations involving multiple sensing domains, the valence (the number of charges that move through the membrane in a single sensor unit) can be changed without noticeably altering the slope of the activation curve. Furthermore, if the altered S4 segment does not contribute significantly to the voltage sensing mechanism, relying on shifts in the slope of the activation curve to detect valence changes can also be misleading. Stuhmer et al. (1989) found that while mutations in the S4 segment of domain I altered the activation properties of the Na+ channel, the equivalent mutations in the second domain had little effect. It is possible that the IVS4 segment of VGCCs also contributes minimally to the gating mechanism and the effect of mutations in this segment are not detectable from the activation curve. A more accurate way to measure the voltage dependence of activation is to analyze the gating currents (reviewed in Bezanilla and Stefani, 1998; and Yellen, 1998). This would allow the detection of very small changes in the activation properties of the channel. Another possibility is that, although the mutation does not alter the voltage sensor of the channel, it does affect coupling of the sensing mechanism to the opening of the channel pore. For example, while the oc1B.6U subunit detects changes in membrane potential normally, it could take longer for the pore to open. It is possible to explore this aspect of channel activation by recording from single channels and measuring the \"first latency\". The first latency is the time between the test pulse and first channel opening; this reflects the time it takes for the channel to go from the closed to the open state once it has sensed the potential change. 203 Given the rather severe phenotype of unc-2(ra611) mutants, it is unlikely that this mutation causes only subtle defects in UNC-2 function. The phenotype of unc-2(ra611) mutants is indistinguishable from that of putative unc-2(null) mutants (Table 9; Figures 19 and 20), suggesting that the G1254R mutation greatly reduces or eliminates UNC-2 function. Intuitively, one would not predict that the consequence of increasing the voltage-sensitivity of a channel would be to lessen its activity; rather, it could be expected that heightened voltage-sensitivity would cause.the channel to open inappropriately upon relatively weak depolarizations. Conversely, it is possible to understand how a delay in channel opening could produce the uncoordinated phenotype of the ra611 animals. For example, increasing the time between the depolarization of the presynaptic membrane and channel opening would delay release of neurotransmitter. It is conceivable that this delay would have profound effects on coordination of body wall muscle contraction, resulting in the kinked phenotype seen in unc-2 animals. A third possibility is that the introduction of the large positively-charged R-group of the arginine residue impedes movement of the channel regions involved in activation gating either electrostatically or sterically. The S2 transmembrane segment contains negatively-charged residues that may provide countercharges to the lysines and arginines in S4 (Papazian etai, 1995; Seoh etai, 1996; Tiwari-Woodruff etai, 1997). The substitution of the glycine with an arginine, thus adding an extra unpaired change, could destabilize the protein or disrupt its insertion in the membrane. We have noticed that whole cell currents recorded from cells expressing the mutant construct are not as large as those from cells expressing the wild-type channel. While this may be an artifact of the transfection procedure, it could also reflect a defect in the cellular processing or membrane stability of the mutant channel protein. For example, Papazian et al. (1995) found that neutralization mutations in the S4 regions blocked maturation of the Shaker protein, suggesting that they prevented proper protein folding. Staining cells transfected with mutant and wild-type cDNA with an anti-oc1B antibody might reveal fewer channels in the membrane and possible 204 staining in intracellular protein trafficking structures such as the Golgi. Alternatively, the larger arginine side chain could make it more difficult for the S4 segment to move through the membrane, rendering the channels essentially non-functional. Presumably, cells expressing the mutant protein would have a similar number of channels inserted in the membrane, but these channels would have a lower probability of opening or would open more slowly. If this is the case, it would be expected that staining cells transfected with the anti-a1B antibody would reveal a similar number of channels in the membrane compared to the wild-type. Concomitantly, single channel recordings could be performed to determine open probability and first latency of the cx1B.611 channel. One other issue that must be taken into consideration when interpreting the ra611 electrophysiology data is the small [but perhaps significant] difference between UNC-2 and the vertebrate VGCC a, subunits. As discussed above, compared to a I B the UNC-2 channel has one fewer charge in the S4 segments in domains LI and IV (Figure 13). There remains the possibility that this structural difference alters the effect of the mutation on channel function. For example, while an increase from five positive charges to six in the IVS4 segment does not appear to alter the electrophysiological properties of the a 1 B channel, the increase from four to five charges may have a substantial impact on the C. elegans channel. General Discussion - Ca 2 + channels in synaptic transmission. UNC-2 is most closely related to the a 1 A and a 1 B P/Q- and N- type VGCC. A wealth of pharmacological studies have linked these two channel types to neurotransmitter release in the vertebrate nervous system (Kerr and Yoshikami, 1984; Dooley et al, 1988; Lundy and Frew, 1988; Dutar et al, 1989; Herdon and Nahorski, 1989; Takemura et al, 1989a; Wessler et al, 1990; Home and Kemp, 1991; Luebke et al, 1993; Potier et al, 1993; reviewed in Dunlap et al, 1995). Behavioral studies suggest that the UNC-2 a, subunit is required for synaptic transmission in C. elegans (E. A. Mathews, this study; also see Miller 205 et al, 1996). In this section, I discuss the process of neurotransmitter release in more general terms, with an emphasis on the role of VGCCs and Ca2+-mediated signaling in this process. In addition, I speculate on the relationship between alterations in channel electrophysiological properties observed in the heterologous expression system and the behavioral defects manifested by unc-2 mutants. A general mechanism of neurotransmitter release. Influx of Ca 2 + through VGCCs initiates the exocytotic release of neurotransmitter. Arrival of the action potential at the axon terminal depolarizes the synaptic membrane activating VGCCs localized at the active zone. Neurotransmitter release occurs within 200 psec of depolarization of the synaptic terminal and requires relatively high concentrations of intracellular Ca 2 + to trigger fusion of the vesicles with the synaptic membrane. Due to the efficiency of Ca 2 + buffering and sequestration mechanisms, the intracellular Ca 2 + concentration drops off rapidly as a function of distance from the point of entry. Thus, the exocytotic machinery must be located close to the Ca2 +, leading to the hypothesis that the vesicles are physically linked to the VGCC complex (reviewed in Catterall, 1999). Current molecular models of excitation-release coupling tend to separate the process into three steps: docking, priming, and fusion (Rand and Nonet, 1997). As the boundary between the events involved in the docking and priming processes is somewhat arbitrary, they will be discussed together. Vesicles synthesized in the neuronal cell body are transported along microtubules down the axon to the synaptic terminal. Upon reaching the terminal, they are attached to the cytoskeleton close to the active zones through synapsin and other proteins. Tethering the vesicles in close proximity to the exocytotic machinery appears to regulate the pool of vesicles available for release. Following an exocytotic event, vesicles from this pool are translocated to the active zone where they are docked, ready for release. The docking process is believed to involve the vesicle membrane protein syntaxin. 206 The priming process involves the formation of a stable interaction between the vesicular and neuronal plasma membranes. This process is thought to occur through formation of a complex comprised of vesicle- (v) and synaptic membrane- (t; target) associated proteins called the SNARES (SNAP receptors). v-SNARES on the synaptic membrane (e.g. VAMP/synaptobrevin) are thought to form a complex with t-SNARES (including syntaxin and SNAP-25). The SNARE complex is believed to serve as a receptor for soluble fusion factors, namely the ATPase, NSF (n-ethylmaleimide-sensitive fusion protein). NSF binds to the SNARE complex, and the stability of the SNARE-NSF structure appears to be regulated by ATP . Upon ATP hydrolysis by SNARE-associated NSF, the NSF is released and the complex undergoes a conformation change which brings the vesicle in close contact with the cell membrane, ready for exocytosis upon the appropriate releasing signal. Membrane depolarization results in the activation of VGCCs at the active zone, and the influx of Ca2 +. Because fusion of the vesicle and synaptic membrane (and concomitant release of neurotransmitter into the synapse) occurs within hundreds of microseconds of membrane depolarization, the docking and priming steps are presumed to occur prior to depolarization. Thus, rather that acting to trigger release per se, the incoming Ca 2 + is thought to release an inhibitory mechanism that prevents premature vesicle fusion. Several candidate proteins have been identified which may act in this capacity. One, synaptotagmin, is associated with the vesicle membrane. Synaptotagmin has been shown to bind Ca 2 +, phosopholipids, and syntaxin, leading to the suggestion that it is the sensor that detects Ca 2 + influx. However, C. elegans and D. melanogaster mutants lacking synaptotagmin retain at least some regulated neurotransmitter release (DiAntonio et al., 1993; Nonet et al, 1993), indicating that there are other proteins involved in this process. A protein encoded by the C. elegans unc-13 gene also appears to be important in neurotransmission, as may be the rab3 GTPase, which is associated with the synaptic vesicle and dissociates upon fusion with the membrane, and the vesicle-associated protein 207 synaptobrevin (reviewed in Sollner etai, 1993; Rand and Nonet, 1997; and Catterall, 1999). Recent work has demonstrated that synaptic vesicles are tethered directly to VGCCs at the active zone through Ca2+-dependent interactions of the SNARE complex, specifically SNAP-25 and syntaxin, with a segment of the II-III loop of N- and P/Q-type channels dubbed the synprint (synaptic protein interaction) site (Sheng et al, 1994; Rettig et al, 1996). Optimal binding of the SNARE complex to the synprint site occurs at Ca 2 + concentrations between 10 and 30 LuM. Synaptotagmin also binds to VGCCs in a Ca 2 + manner competing with the SNARE complex for the synprint site. As Ca 2 + concentration increases above this range, as would occur upon opening of VGCCs, the affinity of the SNARE binding decreases. At the same time, synaptotagmin binding increases, promoting fusion of the vesicle with the membrane (reviewed in Catterall, 1999). The mechanism of neurotransmission is highly conserved. The mechanism of regulated exocytosis appears to be conserved throughout evolution as homologues of many of the proteins involved in the process have been found in yeast, Drosophila, C. elegans, and vertebrates (reviewed in Rand and Nonet, 1997). For example, proteins in the rab3 family of small GTP-binding proteins associate specifically with synaptic vesicle membranes and have been implicated in the secretory pathway involving membrane fusion, rab homologues have been identified in mice (rab3B and rab3C) and C. elegans (rab-3). Furthermore, proteins involved in the rab3 regulatory pathway have been identified in the C. elegans genome. Proteins involved in specific stages of exocytotic secretion have also been identified in C. elegans, either through genetic studies or analysis of the sequence provided by the Genome Sequencing Project (reviewed in Rand and Nonet, 1997). Again, many of these proteins have homologues in other species, suggesting that a similar mechanism of synaptic release is shared by many diverse species. Vesicle transport from the cell body to the 208 synapse is thought to involve a kinesin-related protein that has been identified in both C. elegans (unc-104) and mouse (KEFIA) (Otsuka et al, 1991; Hall and Hedgecock, 1991; Okada et al, 1995). As mentioned above, vesicle attachment to the cytoskeleton is thought to be mediated by synapsins, as well as other proteins. While no synapsin homologues have been identified in C. elegans, another protein encoded by the unc-31 gene, may play an analogous role. UNC-31 localizes to all neurons in C. elegans and is similar to CAPS, a Ca 2 +- and actin-binding protein found in neuroendocrine cells. The Ca2+-binding capabilities of UNC-31 suggest that it may also play a role in the regulation of vesicle fusion in response to the Ca 2 + signal (Avery etal, 1991; Martin, 1994; reviewed in Rand and Nonet, 1997). The unc-64 gene encodes the C. elegans homologue of the t-SNARE syntaxin (Saifee et al, 1998 ). Null mutations in unc-64 result in animals that are paralyzed upon hatching, suggesting an important role in vesicle docking in the C. elegans nervous system, unc-18 was first identified in C. elegans as a vesicle associated protein; these mutants have defects in transmission and accumulate vesicles at the synapse and therefore is believed to be necessary for stable docking of the vesicle at the active zone. UNC-18 is similar to a number of proteins involved in the yeast secretory pathway (Seclp, Slplp, and Slylp) and homologues have been identified in Drosophila (rop) and vertebrate nervous systems (Muncl8, n-Secl, and rb-Secl). UNC-18/Munc-18 appears to mediate the formation of the complex between the v-SNARES and t-SNARES as the protein dissociates from the vesicle upon complex (Hosono et al, 1992; Gengyo-Ando et al, 1993; Hata et al, 1993; Ferro-Novick and Novick, 1993; Harrison et al, 1994). The SNAP receptor proteins involved in the priming process also have homologues in C. elegans. In addition, genes encoding NSF and SNAPs have also been identified in the genome sequence (reviewed in Rand and Nonet, 1997) .Mutants in VAMP/synaptobrevin (snb-1), SNAP-25 (ric-4), syntaxin (unc-64), and synaptotagmin (snt-1) have been identified (reviewed in Rand and Nonet, 1997; Nonet etal, 1998), enabling interactions between these proteins to be studied. For example, syntaxin appears 209 to associate with UNC-18, and synaptobrevin and synaptotagmin may have overlapping roles in exocytosis (Nonet et al, 1993; Nonet et al, 1993; Rand and Nonet, 1997; Nonet et al, 1998). This last finding helps explain the reduction, but not elimination, of neurotransmission in animals lacking synaptotagmin (DiAntonio et al, 1993; Nonet et al, 1993; Littleton etai, 1993). UNC-2 may provide the Ca 2 + signal in C. elegans nervous transmission. Studies examining genetic interactions between proteins involved in synaptic release have not yet identified the specific VGCC responsible for the Ca 2 + influx that triggers exocytosis. Because of the similarity of UNC-2 to the DHP-insensitive channels in vertebrate systems, it is tempting to speculate that unc-2 plays a role in neurotransmission in C. elegans homologous to that of the P/Q- and N-type channels. This hypothesis is strengthened by the mutant phenotype of unc-2 animals, which exhibit the mild locomotory defects and aldicarb-resistance suggestive of an involvement in neurotransmission. I conclude that the ra605, ra610, and md328 alleles are null based on the finding that a, subunits truncated at the beginning of the carboxyl tail do not show measurable expression (T. Stea, personal communication; Wei etai, 1994). The ra611 and ra672alleles both contained missense mutations, suggesting that they may encode functional proteins. However, while this appears to be the case for the ra612 mutation, the ra611 allele seems to be a functional null. The ra612 mutation, a glycine to arginine substitution in the carboxyl tail, was introduced into the rat brain oc1A subunit. Expression in HEK cells resulted in a current that had faster inactivation kinetics compared to the wild-type current. This would have the effect of shortening channel open time, thereby decreasing the amount of Ca 2 + influx. Furthermore, the depolarizing shift in the voltage-dependence of activation results in a channel that requires stronger depolarizations to become activated, and the hyperpolarizing shift in the steady-state of inactivation renders the channel less capable of opening at a 210 given potential than are the wild-type channels. The net effect of these alterations in channel properties is to reduce the magnitude of Ca 2 + influx during the depolarization. In turn, this would reduce the amount of neurotransmitter released. The phenotype of unc-2(ra612) mutant animals, while qualitatively similar to the other unc-2 mutants described in this and other studies, is noticeably less severe. This observation is supported by the quantitative analysis of two well-defined behaviors, locomotion and defecation; ra612 animals were considerably less affected than the null mutants (Table 9; Figures 19 and 20). Thus, the phenotypic data suggests that, not only is the ra612 mutant channel expressed, but that it has at least some degree of function. The waveform of the oc1A_612 current indicates that there is a rapid, but short-lived, increase in intracellular Ca 2 +. Presumably, this Ca 2 + burst is sufficient to increase the local Ca 2 + concentration to the threshold levels (20-50 pM) (reviewed in Catterall, 1999) required to initiate vesicle fusion and transmitter release, but transmission is prematurely curtailed, resulting in the uncoordinated phenotype of unc-2. The case of ra611 is somewhat different. The severity of the unc-2(ra611) mutant phenotype suggests that this mutation renders the channel nonfunctional; unc-2(ra611) mutants were comparable to the unc-2(null) mutants in the locomotion and thrashing assays (Table 9; Figures 19 and 20). However, the electrophysiological data reveal no obvious defects in channel properties (Figure 25). The only potential difference was the smaller currents seen in mutant-transfected cells, which could reflect fewer functional channels in the membrane. If the Ca 2 + influx is too small to withstand the powerful buffering and sequestration mechanisms in the cell, it is possible that the concentration never reaches threshold and vesicle fusion would never be effected. Thus, this would yield a null phenotype. 211 Conclusion and future studies. One of the goals of this project was to establish a model system that would help define functional domains of the VGCC a, subunit protein. By combining genetic, molecular, and electrophysiological techniques, I have isolated a number of mutations in the C. elegans UNC-2 protein and examined the functional significance of two of them by introducing them into a vertebrate homologue for study in a heterologous expression system. This system still has a number of drawbacks, however. The molecular manipulation of a, subunit cDNAs, which is technically difficult due to their large size and repetitive structure, is still a necessary aspect of this approach. The functional expression of the UNC-2 VGCC would facilitate the analysis of unc-2 mutations. For example, mutant constructs could be generated much more easily by directly inserting segments of genomic DNA or reverse transcribed cDNA from the mutant animal into the UNC-2 cDNA clone. This would eliminate the need for the manipulations required previously in generating mutant a, clones. Another consideration when using surrogate systems to study structure-function relationships is that in vitro expression systems cannot be expected to mimic all the attributes of the native cell and that any differences may lead to aberrant results. There are at least two examples in the literature of situations in which the expression system has impacted the behavior of the VGCC under study (see Qin et al, 1997; Bourinet et al, 1999). However, one must not overlook the advantages provided by the use of surrogate expression systems in the study of a biological process. Such systems provide a controlled environment that allows for the comparison of members of a family of proteins [the different classes of a, subunit, for example]. In addition, the cells in which the protein is expressed are isolated from others, and are therefore not subject to modulatory input that may impact the cell in its native environment. When this project was begun, electrophysiological recordings from C. elegans neurons had not yet been demonstrated. In the intervening years, techniques have 212 been developed for whole-cell patch-clamp recordings from intact neurons and muscle (Lockery and Goodman, 1998; Richmond and Jorgensen, 1999). To make use of this new technique, and to fully understand the role of UNC-2 in C. elegans physiology, it will be necessary to determine its expression pattern. While I was unsuccessful at localizing UNC-2 using a polyclonal antibody, it is possible to localize proteins using GFP-reporter constructs. Generation of a unc-2: :GFP promotor construct or a functional unc-2::GFP fusion would be useful in this respect. However, in the meantime, the defecation defects of unc-2 mutants strongly suggest that the channel is expressed in the AVL and DVB neurons. Potentially, recordings could be performed on these neurons to examine the effects of mutations in unc-2 in vivo. We have made use of C. elegans as a tool with which to study VGCCs. The approach taken in this work complements the techniques currently used in the field. The ability to generate a wide range of mutations in a gene provides insight into the role of that protein in physiology and behavior. Furthermore, the amenability to molecular manipulation enables the researcher to easily determine the nature of the mutation and its effects on the protein's function. Screens can be designed that would allow one to dissect a known function of the protein, such as its role in neurotransmission or cell migration. Alternatively, one can focus on a region of the protein to elucidate its role. In this study, we found that the ra612 mutation, a single amino acid change in the carboxyl-terminal region, has profound effects on multiple electrophysiological properties of the UNC-2 and rat brain a 1 A VGCCs. 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