ALTERNATE C1 SECRETORY PATHWAYS IN CYSTIC FIBROSIS

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DESCRIPTION: The main goal of the proposed research is to understand at the molecular, immunological, and physiological level the mechanisms and regulation of ion permeation through conductive Cl- channels present in the apical membrane of secretory epithelia, and to elucidate their potential interactions with the cystic fibrosis transmembrane conductance regulator (CFTR). Studies of epithelial Cl- channels other than CFTR have been limited, largely because of the lack of important targets for pharmacological therapy in cystic fibrosis (CF). This laboratory has successfully isolated, purified, and reconstituted a 140 kDa protein from bovine trachea that functions as a DIDS-sensitive anion channel. The application has two specific aims: (1) to test directly the hypothesis that the native 140 kDa protein purified from bovine trachea functions as a regulated, calcium-activated Cl- channel (CaCC). These investigators will examine certain biochemical characteristics of the protein including the extent of glycosylation, its ability to be phosphorylated by protein kinase A (PKA), protein kinase C (PKC), tyrosine kinases (TK), and Ca2+/calmodulin-dependent protein kinases (CaMK), as well as the functional consequences of these phosphorylation reactions in planar lipid bilayers. Other biophysical properties of native and biochemically modified channels such as ion selectivity, pharmacological inhibition, and kinetics will also be determined; (2) to identify and characterize full-length cDNAs corresponding to the polypeptides comprising the 140 kDa Cl- channel of bovine trachea in order to verify that this protein indeed functions as an ion channel, and to identify a human cDNA homolog. The investigators will examine the biochemical and functional expression of the protein encoded by the cDNA expression libraries, and analyze immunopurified protein from the point of view from its biosynthesis. These studies will further our knowledge of the physiological, biochemical, and molecular biological properties of these important Cl- transport pathways and increase our understanding of fluid secretion across airway epithelia so that potential avenues of alternate therapy in CF can be devised and evaluated.