Mutations in a single protein, a cAMP regulated Cl channel referred to as the cystic fibrosis transmembrane conductance regulator (CFTR), are responsible for the cystic fibrosis (CF) phenotype. The disease CF is characterized by thickened airway secretions due, in part, to reductions in basal and cAMP activated Cl secretion. In addition, Na reabsorptive rates in airway epithelia are enhanced two- to three-fold in CF and likely contribute to the decrease in water content in airway fluids. The increased Na movement across airway epithelia is a result of activation of apical membrane amiloride-sensitive Na channels, although it is unclear why mutations in Cl channels lead to functional changes in Na channels. The recent finding that the Xenopus epithelial cell line A6 has both cAMP regulated Cl channels (which are likely CFTR) and Na channels provides a unique model system to examine interactions of CFTR and epithelial Na channels with the electrophysiologic and biochemical tools previously used to characterize Na channels in this cell line. Antisense oligonucleotides which are complementary to CFTR mRNA can be utilized to deplete cells of CFTR mRNA and to inhibit anion transport, as has been demonstrated in lymphocytes and sweat duct cells. The proposed studies will utilize specific oligonucleotides which are complementary to Xenopus CFTR mRNA to deplete CFTR from A6 cells, which will be confirmed by monitoring cAMP activated Cl secretion, levels of CFTR mRNA, and expression of CFTR protein. Subsequent changes in Na channel function will be examined by measurement of amiloride-sensitive transepithelial Na transport. Anti-Na channel antibodies will be used to examine Na channel structure, localization, and biosynthesis. In parallel studies, Dr. D. C. Eaton will examine characteristics of single Na channels with the patch clamp technique. If changes in Na channel function or regulation are observed, CFTR levels will be restored by placing cells in media free of oligonucleotides. Resultant changes in electrophysiologic and/or biochemical properties of Na channels will be assessed. These proposed studies should define changes in the function, structure, or distribution of Na channels which might occur in CF and contribute to the pathogenesis of CF.
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