Na+, K+-ATPase (Na pump) is the target receptor for the biological actions of the digitalis. The precise mechanism and the detailed knowledge of which amino acid side-chain groups are responsible for digitalis binding have remained open questions. The focus of this proposal is to identify the region and the specific amino acids that participate in digitalis binding to the Na+, K+-ATPase and are essential for ion transport. The strategy of the study is to use native digitalis and its derivative with the complementary technology involving protein chemistry and molecular biology to provide direct evidence of a specific, covalently bound intermediate (enzyme-digitalis complex), that is undetectable by ordinary chemical analyses. Purified cardiac Na+, K+-ATPase from rabbit and rat will be used for the affinity labeling. The newly identified residues will guide the design of site-directed mutagenesis studies to further characterize and explore the digitalis binding sites in the three isoforms of the rat Na+, K+-ATPase.
Five specific aims are proposed to achieve the objective:
Aim 1, to identify the structural determinants at the digitalis binding pocket of both ouabain-sensitive (rabbit) and ouabain-resistant (rat) Na+, K+-ATPase, and to test the hypothesis that the newly identified drug binding sites are the universal digitalis binding sites at the protein level;
Aim 2, to characterize the newly identified amino acids that reside in the digitalis binding sites at the molecular level, and to further test the hypothesis that these sites are the universal drug binding sites for three isoforms of the Na+, K+-ATPase;
Aim 3, to determine whether the newly identified amino acids contribute to the ouabain-sensitivity,and to test whether the new drug binding sites are structurally near the substrate binding site in the extracellular domain of the Na+, K+, ATPase;
Aim 4, to explore the functional relationship between the neighboring residues and the newly identified amino acids in the same domain of the enzyme, and to test the hypothesis that these surrounding residues may play important roles in regulating the drug-sensitivity of the Na+, K+-ATPase and the binding of substrates and digitalis;
Aim 5, to examine the interactive role of residues between other extracellular domains (H1-H2 & H3-H4) and the newly identified domains, and to further explore the three-dimensional nature of the digitalis binding pocket. These studies will provide fundamental information to gain insight into the nature of the digitalis-receptor reaction mechanism. The ultimate goal of the research is to obtain new knowledge that will allow us to elucidate the structure-function relationship of the enzyme for a better understanding of the biological processes mediated by the Na+, K+-ATPase in health and disease.
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