Our long-term objectives are to determine the biological roles, structure-function relationships, and genetic regulatory mechanisms for members of the H,K-ATPase family of enzymes. H,K-ATPases are ATP-dependent pumps that mediate the secretion of acid in stomach, absorption of potassium in colon, and both acid secretion and potassium absorption in kidney. Thus, they play essential roles in the digestion of food and in the maintenance of both acid-base balance and potassium homeostasis. A better understanding of the biological functions of these enzymes, the genetic and physiological mechanisms controlling their expression, and their structure-function relationships should lead to improved treatment for a variety of gastrointestinal and kidney diseases. We recently isolated and characterized cDNAs encoding the distal colon H,K-ATPase alpha-subunit and have obtained evidence demonstrating that it is expressed in kidney and suggesting that it may correspond to the H,K-ATPase of the distal nephron. The gene for the distal colon H,K-ATPase alpha-subunit will be analyzed in order to obtain basic information needed for gene regulation studies, and a cDNA encoding its beta-subunit will be isolated and characterized. PCR and genomic cloning studies will be performed to identify the rat homolog of the human alpha-D gene, which seems to encode a third distinct H,KATPase isoform. Changes in distal colon H,K-ATPase mRNA and protein levels in response to alterations in acid-base balance, potassium depletion and hormone treatment will be analyzed by Northern blot and immunological techniques. The cell-type specificity and membrane location of the enzyme will be determined by in situ hybridization and immunocytochemistry. Embryonic stem cell technology will be used to prepare a mouse in which the distal colon H,K-ATPase gene has been disrupted, thereby allowing an evaluation of the physiological effects of the absence of this gene, which would be expected to include disturbances in acid-base balance and potassium homeostasis. The regulatory elements of the genes encoding the alpha and beta subunits of the gastric H,K-ATPase and the alpha subunit of the distal colon H,K-ATPase will be identified using transgenic mice, DNA footprint analysis and mobility shift assays. Structure-function relationships of both the gastric and distal colon H,K-ATPases, with particular emphasis on the domains involved in ion-specificity and binding of drugs such as omeprazole and SCH28080 will be examined using H,K-ATPase/Na,K-ATPase chimeras and site-directed mutagenesis techniques.
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