The long-term goal of this project is to determine the structure-function relationships and kinetic characteristics of members of the mammalian K+ transporting ATPase family: the Na,K-ATPase, the gastric H,K-ATPase, and the distal colon H,K-ATPase. The gastric H,K-ATPase and Na,K-ATPase are the pharmacological targets for the treatment of a number of diseases, including gastrointestinal disorders and congestive heart failure. The distal colon H,K-ATPase participates in potassium absorption and acid/base balance in the kidney and colon. It is a potentially important drug target because it can be inhibited by agents that inhibit both the Na,K- ATPase and the gastric H,K-ATPase. A better understanding of the function and structural contributions to function of these enzymes should lead to improved treatment of a variety of human disorders. The cDNAs for the catalytic subunits of the gastric H,K-ATPase, three isoforms of the Na,K- ATPase, and a potential distal colon H,K-ATPase have been isolated and characterized. The predicted amino acid structures of these ATPases are very similar, yet the drug sensitivities differ dramatically. In addition, characterization studies reveal that the gastric H,K-ATPase and the Na,K- ATPase proteins differ in other important biochemical parameters. Molecular techniques and exogenous expression will be combined to analyze H,K-ATPase/Na,K-ATPase chimeras. These studies are designed to define structure/function relationships of these two proteins, with a marked emphasis on regions involved in ion specificity and drug binding. In preliminary studies, an active, ouabain resistant H,K-ATPase/Na,K-ATPase chimera has been expressed in HeLa cells and partially characterized in isolated membranes. Expression studies will also be used to confirm the role of the putative distal colon H,K-ATPase cDNA as encoding a proton/potassium ATPase and to characterize this ATPase. The requirements of the H,K-ATPases for a beta-subunit will be investigated with an oocyte expression system.
