Proton-potassium adenosine triphosphatases (H+, K+-ATPases) located in the renal collecting duct are involved in maintenance of blood K+ balance. Although aldosterone is known to be one of the principal regulators of K+ homeostasis and urinary acidification, no firm linkage has been established between aldosterone and the regulation of genes encoding the subunits of the H+, K+-ATPase or the activity of the pump in the kidney. The long term goal of our research is to gain an understanding of the regulation of H+, K+-ATPase activity in the kidney. At least three H+, K+-ATPases containing distinct isoforms of the alpha subunit populate the collecting duct. These H+, K+-ATPases contain the """"""""gastric"""""""" 1, the """"""""colonic"""""""" 2a or the novel 2b subunit. The 2b H+, K+- ATPase appears to be a candidate for the aldosterone-responsive form of the pump. We propose to study the expression of the 2a and 2b H+, K+- ATPases following two parallel lines of investigation. First, aldosterone-responsive transcriptional regulation of the 2b subunit will be investigated in vivo in a rabbit cortical collecting tubule cell line (RCCT-28A). This will be accomplished by characterizing the response of the 2a and 2b mRNAs, protein levels and H+, K+-ATPase activity following application of aldosterone to RCCT-28A cells. The elements governing transcription from the 2 gene will be located by DNase I hypersensitivity and promoter deletion analysis. The response elements will them be mapped to single-nucleotide resolution by in vivo footprinting studies. Second, the activity of the two 2 H+, K+-ATPases will be studied by expression of the cloned cDNAs in mammalian cells. A direct comparision will be made between the activities and pharmacological properties of the expressed 2a and 2b H+, K+-ATPases. Finally, phosphorylation of the 2 subunits will be investigated as a possible mechanism for differential regulation of the 2a and 2b H+, K+- ATPases.
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