The central goal of this project is to isolate and characterize the apical membrane Cl channel of secretory epithelial cells. The secretory C1 channel represents the rate-limiting step for secretion across airway eptihelia and its regulation by cAMP_-mediated second messenger pathways has been shown to be defective in cystic fibrosis(CF). We will use a secretory epithelial cell line (T84) as the source of Cl channel proteins. Protocols for the solubilization and reconstitution of functional Cl channels will be developed. The approach towards isolation and purification of the transport complex is based on the use of disufonic stilbenes (DS) as chemical probes. Disulfonic stilbenes have been shown to cause a reversible blockade of the secretory Cl channel when incorporated into planar lipid bilayers. We will used this reversible interaction to isolate the Cl channel by standard biochemical techniques. DS-binding proteins will be separated from apical membrane proteins by affinity chromatography on a DS-affinity column. The ability of the DS-binding proteins identified by this approach to mediate conductive anion transport will be accessed using an 1 2 5I uptake assay. The single channel properties of a candidate channel protein will be accessed after incorporation into planar lipid bilayer and compared to the known properties of the secretory Cl channel. Structure-activity studies with various DS-derivatives as well as antibodies to the DIDS-binding site will be conducted to probe the nature of the DS-C1 channel binding site. This approach will permit us to identify the Cl channel and its regulatory subunits. The results will provide us with the necessary tools to achieve a molecular understanding of the secretory cell Cl channel, which underlies electrolyte secretion across the cells lining the airways as well as a variety of other secretory tissues. A molecular understanding of the Cl channel will ultimately permit identification, and possible manipulation, of the components essential for its conduction and gating.
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