The long-term goal of this project is to elucidate the role of the product of the cystic fibrosis (CF) gene, the CFTR, in normal physiology and in the pathogenesis of the disease. The CFTR was recently cloned by reverse genetics and provides researchers the first direct insight into the molecular basis for the disease. The predicted protein has significant homology to an ancient gene family of proteins which couples ATP hydrolysis to transport, confirming the long-held assumption that the protein is involved in transport, but not illuminating its substrate. The specific objectives of the research proposed herein are (1) to determine the subcellular distribution of the CFTR protein, to characterize the kinetics of its biosynthesis and processing in normal and CF cells; (2) to establish cell lines which overexpress the CFTR protein and cell lines in which CFTR expression has been specifically ab- rogated; (3) to investigate the biochemical and physiological properties of such mutant cell lines. We propose to realize these objectives by molecular, immunological and physiological characterization of the above-mentioned cell lines. Specifically, we will study the effects of deletion, overexpression and site-directed mutagenesis of the CFTR on cellular and transepithelial anion transport properties. Lastly, we will attempt to identify potential substrates for the CFTR which may, indirectly, mediate its pathogenic effects.
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