Voltage-gated chloride channels belonging to the CIC gene family have recently been identified as important molecular components of various physiological processes including sarcolemmal excitation, cell volume regulation, organellar acidification, and renal epithelial C1-transport. In mammalian genomes, there have been 9 distinct isoforms identified through molecular cloning, and three of the genes encoding different human CIC channels are responsible for distinct inherited diseases. Despite the clear importance of certain CIC channels, most isoforms have no known physiological function in mammalian tissues. This stems largely from the difficulty in studying these channels in complex organisms. In the complete C. elegans genome, six predicted coding sequences have been identified that exhibit homology with the mammalian CIC gene family. This Project seeks to characterize the primary structure, function, tissue localization, and physiological role of CIC gene family. This Project seeks to characterize the primary structure, function, tissue localization, and physiological role of CIC chloride channels in C. elegans using an integrated approach that utilizes molecular, electrophysiological, and genetic techniques. This comprehensive analysis of the role of CIC channels in such a well characterized model organisms presents unique opportunities to gain insight into the fundamental biological roles played by CIC channels. We will begin with cDNA cloning, functional expression, and determination of tissue localization of each gene (Specific Aim 1). We will next characterize the phenotypes associated with targeted gene disruption of each CIC channel in the work (Specific Aim 2) and be prepared to exploit genetic screens to help identified important interactions of CIC channels with other genes. The latter experiments may reveal the existence of accessory subunits or other protein-protein interactions important for channel function. Lastly, in Specific Aim 3, we have outlined a strategy to determine if heteromultimeric assemblies of CIC channels occur in vivo, and to understand how this impacts on the functional diversity and physiological importance of CIC channels in a multicellular organism.
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