MOLECULAR ANALYSIS OF NA+/K+-ATPASE

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The objective of this proposal is to apply the techniques of molecular genetics to the analysis of Na,K-ATPase structure, function and biogenesis. We have developed an expression system to test the biological activity of cloned rodent Na,K-ATPase genes. This system forms the framework for experiments designed to analyze structure function relationships for the ATPase and control mechanisms underlying ATPase biosynthesis and assembly. The specific aims of the proposal include: 1) Interaction of the Na,K- ATPase with Cardiac Glycosides. We will evaluate the relationship between enzyme structure and the binding of cardiac glycosides. Initial experiments will involve the construction of chimeric cDNA molecules between cDNA molecules encoding ouabain resistant and ouabain sensitive forms of the Na,K-ATPase in order to delineate regions of the alpha chain which interact with the drug. We will then use site directed mutagenesis to alter specific residues in a cDNA encoding a ouabain sensitive ATPase in an effort to convert the encoded enzyme to ouabain resistance. 2) Subcellular Localization of Na,K-ATPase Isoforms. The technique of in situ hybridization will be used to learn whether alternative forms of Na,K-ATPase mRNA are expressed within the same or different cells of a particular tissue. Peptide derived antibodies specific for each ATPase isoform will be developed and used to distinguish alternative ATPase isoforms in cells synthesizing more than one isoform and to identify sites of subcellular localization. The goal of these experiments will be to obtain basic information regarding the relationship between isoform expression and function. 3) Control Mechanisms Affecting Na,K-ATPase Biosynthesis. We will attempt to isolate and characterize DNA sequences which may be responsible for the tissue specific and developmentally regulated expression of Na,K-ATPase mRNAs. Initial interest will focus on identification of the promoter and other 5' control regions involved in the regulation of Na,K-ATPase mRNA expression. We will also attempt to identify regions within alpha and beta subunit mRNAs which may play a role in the postranscriptional regulation of subunit biosynthesis. 4) Relationship of the beta Subunit to Na,K-ATPase Function. We will attempt to develop an assay system which measures the biological activity of the 8 subunit by creating a system in which overexpression of the beta subunit is required for cell viability. This system will then be used to assess structure function relationships for the beta subunit.