Na,K-ATPase is commonly considered as the only specific target for cardiac glycosides used to treat congestive heart failure and cardiac arrhyythmias. Recent characterization of the human ATP1AL1 gene, and functional expression of this gene product, have indicated that the encoded protein (Atp1a11) is a catalytic subunit of a hitherto unknown human H,K-ATPase, which is sensitive to cardiac glycosides; and therefor, may represent a new type of human receptor for these drugs. The identification the Atp1a11 counterpart (the beta-subunit) and the isolation of the entire human ouabain-sensitive H,K-ATPase are proposed in Specific Aim 1. This will allow the characterization of the functional properties of this ion pump by expression of the subunit cDNAs in the oocytes of Xenopus laevis and in insect cells for study of transport functions and enzymatic properties, as proposed in Specific Aim 2. The discovery of this novel human ATPase has substantially expanded the possibility for more detailed characterization of structure-function relationships of different groups of ion transporters forming the X,X- ATPase family. We plan to generate chimeric constructs, in which intramembrane segments and extracellular loops of the Atp1a11 are replaced in different combinations with the corresponding fragments of Na,K-ATPase and gastric H,K-ATPase, and analyze their enzymatic and transport properties using heterologous expression systems (specific Aim 3). This will allow us to define the roles of particular intramembrane segments in ion specificity and inhibitor sensitivity of different X.K-ATPases and to identify those that are in direct contract with beta-subunit. To elucidate major parameters of intramembrane structure of X,K-ATPases-the orientation of transmembrane segments relative to each other and to the lipid bilayer, in Specific Aim 4, we propose direct probing of the intramembrane structure by chemical modifications of the Na,K-ATPase which is the most accessible for the study on the protein level. Topological studies (Aims 3,4) will provide considerable insight into the spatial structure of membrane moieties of X,K-ATPases. The anticipated results of the proposal in whole, will be a fundamental advance in our understanding of structure- function relationship of the X,K-ATPases: the ouabain-sensitive Na,K-and H,K-ATPases and obabain-resistant gastric H,K-ATPase.
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