The central goal of this project is to define the role of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in C1 secretion by epithelial cells. Established cell lines and cells expressing wild-type and mutant forms of CFTR will be used to study the normal functions of CFTR and the way in which its functions are modified by disease mutations. Whole-cell current and single-channel methods will be used to define the regulation of the CAMP-activated C1 conductance, G(CAMP), whose stimulation is defective in secretory epithelial cells from cystic fibrosis patients. The interaction of C1 channels with phosphatases, proteases and cytoskeletal elements will be examined in an attempt to identify the regulatory factors responsible for the loss of channel activity in excised membrane patches. Agonist-induced changes in membrane capacitance will be determined to see if fusion of internal membrane vesicles with the surface membrane accompanies the activation of G(CAMP). The mediators of the direct activation of apical C1 channels by agonists that act from the mucosal solution will be identified. The properties and regulation of C1 conductances in absorptive sweat duct cells and those in secretory cells will be compared. We will combine electrophysiology with CFTR expression to determine the cell biology and structure-function relations of CFTR. A technique for tagging CFTR with a small peptide sequence will be developed for its identification by Western blotting, immunoprecipitation and immunofluorescence. This approach will be used to study the dose-response relation, cellular location an membrane topology of expressed CFTR by introducing the sequence into various sites. We will continue collaboration with investigators at the University of Michigan to determine the effects of functional domain modifications and natural mutations on G(CAMP) in airway and pancreatic cells. These studies will identify CFTR's normal cellular functions and the way in which they are impaired by disease mutations.
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