ALTERNATE CHLORIDE ION SECRETORY PATHWAYS IN CYSTIC FIBROSIS

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The main goal of the proposed research is to understand the mechanisms and regulation of ion permeation through C1- channels other than the cystic fibrosis transmembrane conductance regulatory (CFTR) present in secretory epithelia. These alternative C1- channels may be useful and important targets for pharmacological therapy in cystic fibrosis (CF). Our laboratory has successfully isolated and cloned a protein from bovine trachea that behaves as a Ca2+ -sensitive C1- channel (CaCC), and has semi-purified and reconstituted an outwardly-rectified C1- channel (ORCC). This application has four specific aims: (1) to test the hypothesis that the translated bovine tracheal cDNA forms an anion channel of identical characteristics to the native protein, that the 38 kDa subunit of the native tracheal CaCC protein is the result of post- translational processing of the cloned 100 kDa CaCC cDNA product, and to determine the biochemical properties of both native and cloned CaCCs. The functional properties of the proteins will also be characterized following reconstitution into planar lipid bilayers or transfection into eukaryotic cells; (2) to identify a full-length cDNA corresponding to the human CaCC homolog and to characterize the translated protein. The molecular structure and function of the human homolog of the bovine CaCC will be determined by screening of appropriate human epithelial cDNA libraries; (3) to purify a protein that behaves as an ORCC from bovine tracheal apical membrane vesicles and to identify and characterize the full-length cDNA that encodes this protein. Candidate proteins will be used to raise polyclonal antibodies that will be used to screen a bovine tracheal cDNA expression library. The ultimate goal is to isolate a full-length cDNA that encodes an ORCC and to characterize the translated protein with the aim of understanding is potential interaction with CFTR and/or other ion channels; (4) to determine if heterologous intestinal specific expression of the CaCC can overcome the lethal intestinal obstruction found in the CF knockout mouse model. We will test the hypothesis that tissue specific expression of the bovine CaCC in the intestine will prevent the lethal consequences of intestinal obstruction by ameliorating the adverse effects of impaired chloride secretion in the intestine. These studies will further our knowledge of the physiological, biochemical, and molecular properties of these important C1- transport pathways and increase our understanding of fluid secretion across airway and intestinal epithelial so that potential avenues of alternate therapy in CF can be devised and evaluated.