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Molecular and genetic analysis of the UNC-2 voltage-gated calcium channel in Caenorhabditis elegans Mathews, Eleanor Alexandra


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 α₁ 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. 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 at 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 α₁[sub A] (P/Q-type) and α₁[sub 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 voltagedependence 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

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