Ionized calcium is unique as a second messenger in that it cannot be metabolized and hence cells must regulate intracellular levels though an array of storage compartments and specialized binding and gating proteins. The molecular nature of many of the Ca2+ channels identified by physiological approaches remains unknown (1). The sequencing of the C. elegansgenome has added a new resource into the investigation of the molecular basis of Ca2+ signalling. Analysis of the C. elegans genome sequence has identified three families of proteins distantly related to the TRP family of cation channels. TRP channels are thought to gate Ca2+influx in response to Ca2+ store depletion and/or other signalling molecules (2). Of these families, the MGCs (Melastatin and GON-2 like channels) are widely distributed and expressed in nature with several homologues in man, Drosophila and mouse. In C. elegans, there are 3 members of the MGC family:
gon-2, which is required for gonadogenesis (3 and E Lambie, pers comm.) and two further channels, C05C12.3 and F54D1.5. Like TRP channels, MGCs show similarity to the pore regions of voltage gated Ca2+ channels. At the amino acid level they are approximately '-24% identical to TRP channels whilst identity to each other and human homologues is 36-40%. Our working hypothesis is that they are Receptor Activated Ca2+ Channels (RACCs) and/or Store Operated Channels (SOCs). We are currently characterising C05C12.3 and F54D1.5. Using 5 RACE we determined the 5 ends of the mRNAs. We cloned the full length cDNAs of C05C12.3 and F54D1.5. This information was used to design GFP fusion constructs to determine expression patterns. Confocal microscopy shows that both proteins are strongly expressed in the intestine and F54D1.5 is also expressed in head neurons. In situ hybridization of C05C12.3 confirms this result. The expression pattern for
gon-2 has not yet been determined (E Lambie, pers comm.). Currently we are using RNAi to specifically decrease the expression of the MGC genes in order to further characterise the function of these proteins. References: 1) Parekh A. B. and Penner, B. (1997) Physiol. Rev. :902-930 2) Barritt, G. J. (1999) Biochem. J. 337:153-169 3) Sun, A.Y. and Lambie, E. J. (1997) Genetics g:1077-1089