The main goal of the proposed research is to elucidate the molecular basis of operation of the conductive sodium entry mechanism that exists in the outer (or apical) plasma membrane of all electrically high resistance epithelia. This sodium entry step is an essential component of the overall active transport system responsible for the net movement of salt and water across these tissues. This entry process is passive, is regulated hormonally, and is sensitive to inhibition by the diuretic drug amiloride.
The specific aims fall into three categories. First, studies will be done to characterize both biochemically and physiologically the isolated and purified Na+ channel protein. These studies will entail improvements in the isolation and purification scheme, determination of subunit number, molecular weight, carbohydrate composition, and location of amiloride binding site(s). Investigations of whether the channel protein can be phosphorylated or methylated, both in vitro and in vivo, will also be undertaken. Second, studies will be done to analyze the kinetic characteristics of the Na+ entry channel, utilizing reconstitution procedures to measure single channel properties in planar lipid bilayers and in lipid vesicles. These experiments will elucidate the cation selectivity of the channel, the nature, the sidedness, and the voltage-dependence of the amiloride block, and whether phosphorylation or methylation of the channel protein per se has any functional consequence. Third, monoclonal and polyclonal antibodies directed against the purified Na+ channel protein will be prepared. These antibodies will be used to build an immunoaffinity column to improve further the channel isolation protocols, and as probes to localize channel protein in different cell types (cultured A6 epithelia and rat cortical collecting tubule). These studies will contribute to our knowledge of the kinetic, biochemical, and physiological properties of this ubiquitous transport system, and increase our understanding of the mode of action of amiloride and other diuretic compounds.
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