Worm Breeder's Gazette 13(3): 78 (June 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have previously identified and cloned a family of five distinct types of voltage-gated calcium channels expressed in the mammalian central nervous system (Snutch and Reiner, Current Opinion Neurobiol. 2:247-253, 1992; Soong et al, Science 260:1133-1136, 1993). In addition to current structure and function studies examining exogenously expressed cloned mammalian calcium channels, there is a need to define the physiological roles of calcium channels using genetic model systems. The correlation of mutant phenotypes with defined alterations that affect electrophysiological and pharmacological properties may identify important structural components that might not be predicted using site-directed mutagenesis approaches. In addition, the genetic and molecular analysis of extragenic suppressors of calcium channel mutants will both define important interactions between calcium channel subunit proteins and also identify novel gene products that interact with the calcium channel complex. As a first step towards these goals, we have used degenerate primers homologous to cloned neuronal alpha subunits and the polymerase chain reaction to amplify RNA from C. elegans. The resulting 327 bp fragment was used to screen the YAC grid and under high stringency conditions we identified three overlapping YACs ( Y76F7 , Y23A3 and Y5E3 )that hybridized to the probe. Subsequently, eight cosmids underlying the YACs were screened and the PCR fragment ( pCe2 )localized to a single cosmid ( T02C5 ).These experiments localize a calcium channel alpha subunit gene to the left arm of the X chromosome between unc-2 and dpy-3 .Sequence determination of an ~ 2 kb portion of the cosmid and several cDNAs isolated from the Barstead library identify an ancestral alpha subunit that shares between 41% and 65% amino acid identity with cloned rat brain neuronal al subunits. High stringency genomic DNA and YAC blot analyses suggest that pCe2 represents a single copy element in the worm genome and does not react with loci previously identified as representing two other calcium channel alpha subunit genes ( egl-19 on LG IV, Lobel et al, WBG 13(2):71 and on LG II, YAC Y24H1 between fer-15 and rol-6 ,Lobel et al, WBG 13(1):46). The results suggest that similar to mammals, C. elegans possesses a diverse family of voltage-gated calcium channels. further experiments are underway to more precisely map the X-linked al subunit gene and to generate polyclonal antibodies for localization studies. In addition, a full length cDNA will be isolated in order to determine the electrophysiological and pharmacological properties of the channel upon exogenous expression in Xenopus oocytes.