MOLECULAR AND PHYSIOLOGIC STUDIES OF A COLONIC K+ ATPASE

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This application proposes continuation of studies of potassium transport that were first initiated in 1975. The present proposal plans the molecular and physiologic characterization of the K-ATPase present in the rat distal colon. Active potassium absorption has been identified in this epithelium and is enhanced by both aldosterone and dietary potassium depletion. The PI has proposed that its cellular mechanism is a K-H exchange energized by a novel K-ATPase. The PI has recently cloned a cDNA from distal whose deduced amino acid sequence appears to represent a novel a subunit P-type ATPase. This proposal plans studies to express this cDNA in transfection studies with a suitable colon cell line to determine whether this cDNA encodes the putative K-ATPase; as P-type ATPases frequently require both alpha and beta subunits for full expression it may be necessary to initiate studies to clone the beta subunit of this putative K-ATPase. Antibodies to synthetic segments of the deduced amino acid sequence of this cDNA will permit identification of the K-ATPase protein immunochemically and to examine its cellular distribution. We plan to continue in situ hybridization studies to establish the cellular distribution of its message. We also plan to determine the regulatory control of the enhancement of K-ATPase by aldosterone and/or potassium deprivation by examining their effects on its message, protein, and enzyme activity. These experiments will also establish the cellular mechanism of potassium movement across apical and basolateral cell membranes. In these proposed studies we must identify appropriate cell lines for the expression and regulation studies; and we plan to determine potassium transport primarily by both apical and basolateral membrane vesicles.