Our long-term objective is to characterize the mechanism by which CFTR controls membrane recycling and C1- conductance in reabsorptive sweat duct cells and in the heterologous expression systems. A major premise of this proposal is that CFTR regulates membrane recycling which, in turn, influences the cell surface expression of C1- channels and/or proteins that regulate these channels. Optical and electrophysiologic techniques will be used to achieve the following specific aims: 1) to relate alterations in CFTR expression to changes in endocytosis/exocytosis and C1- conductance in these experimental systems; 2) to provide a detailed characterization of the C1- conductance properties of reabsorptive sweat duct cells; 3) to characterize the regulation, ultrastructure and dynamics of the endocytic and exocytic pathways in these systems, and the identities of those vesicles whose movements are CFTR-dependent: 4) to immunolocalize CFTR in these systems and relate the location of this protein to those vesicles whose movements are CFTR-dependent and 5) to characterize the mechanism by which CFTR directs exocytosis in these systems and test the hypothesis that this exocytic process causes the CFTR-dependent upregulation of C1- conductance. Our results should provide significant insights into the mechanism by which the CF gene product controls epithelial C1- transport and membrane turnover; insights that may prove useful for designing treatments for this disease.
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