Chloride channels play key roles in epithelial ion transport in cells and in regulation of cell volume. However, little is known about the molecular mechanisms of chloride-channel function and regulation. The cultured amphibian renal-cell line A6 is a uniquely suited model for such studies because these cells are well differentiated and have robust, hormonally regulated, sodium and chloride transport mechanisms. At present we lack sufficient knowledge about the structures responsible for gating of epithelial chloride channels, their membrane localization, and the mechanisms for modulation of channel activity by hormones and intracellular messengers. Dr. Wills will use PCR and expression-cloning methods to isolate, sequence, and functionally express chloride channels from A6 epithelium. In preliminary studies, she has shown that chloride channels similar to the ClC family and pICln are present in these cells. Dr. Wills will obtain full-length clones for these proteins and assay their channel properties from whole-cell current or single-channel measurements following expression in Xenopus oocytes or in insect (SF9) cells. The results of these studies will contribute to basic knowledge of epithelial chloride-channel function and will allow characterization of two novel ClC chloride channels. The unique features of A6 amphibian renal-epithelial cells should facilitate the functional expression of these chloride channels and allow the first expression of wild-type clones of osmotically-regulated ClC channels. The project will provide the foundation for future structure-function studies of epithelial ion channels.
