The long term objectives of this research are to advance our knowledge of the molecular mechanism of active transport of Na+ and K+ by NaK-ATPase (the sodium pump); an enzyme of the plasma membrane that maintains the integrity and the excitability of the myocardium, is the receptor for the positive inotropic actions of digitalis drugs, and regulates cardiac genes involved in the hypertrophic growth of the cardiac myocyte. The proposed studies are focused on the recent progress of this laboratory relating the ion transport function of the enzyme to the structures of its transmembrane domains. In experiments of Specific Aim 1, we shall use proteolytically digested and/or chemically modified preparations of the purified enzyme in order (a) to identify the locations of the two distinct cations occulation pockets (the binding sites and their access channels) within different transmembrane helices; and (b) to characterize the properties of the bindings sites and the access channels of each occlusion pocket, and the interactions among the two pockets, by experiments on occlusion-deocclusion kinetics of 86RB+ and 22Na+. Since we have established recently that the catalytic ATP site and the allosteric ATP site are two distinct entities, in studies of Specific Aim 2 we shall first use digested preparations of the enzyme that contain only the allosteric site to identify the amino acid residues involved in this binding site by chemical modification experiments. We shall then alter the identified residues by site-directed mutagenesis, and conduct functional studies on the mutants expressed in insect cells, in order to clarify the postulated roles of the allosteric ATP site in the regulation of the two cation occlusion pockets, and in the turnover of the phosphointermediate. In studies of Specific Aim 3 we shall continue our chemical cross-linking experiment on the digested preparations on the digested preparations of the purified NaK-ATPase to map the three-dimensional packing of the transmembrane helices, and to relate these helix-helix interactions to the functions of the multiple cation occlusion sites and their access channels. These studies will clarify structure-function relationships of an enzyme that is central to the regulation of cardiac contractility and growth in the normal and failing hearts.
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