The Na,K-ATPase, also called the sodium pump, is a protein that is found embedded within the plasma membranes of all animal cells. The function of the Na,K-ATPase is t transport sodium out of the cell, and to transport potassium into the cell. The transport of these ions by the sodium pump occurs against electrochemical gradients for each ion, and the energy for the performance of this work is derived from the enzymatic hydrolysis of intracellular ATP. The Na,K-ATPase is also the only known receptor for digitalis-like drugs. Despite the important role of Na,K-ATPase in animal cell physiology, the mechanism of active transport catalyzed by this protein is unknown. A one approach to the resolution of this problem, this application describes several experiments designed to obtain structural data about Na,K-ATPase. Emphasis is placed on regions of the alpha subunit that are of particular importance for an understanding of ATP-coupled ion transport the ATP binding site, the membrane spanning regions, and the cardiac glycoside binding site. The structure of the beta subunit will also be examined. Photochemical and functional group-specific probes will be used to identify peptides derived from each of these sites, and the amino acid sequences of the peptides wilT be determined. The importance of individual amino acids for the chemical reactions of Na,K-ATPase will also be determined using structurally-modified forms of Na,K-ATPase biosynthetically expressed in the yeast S. cerevisiae. This expression system will enable the structural features of each polypeptide subunit of Na,K-ATPase that are important for membrane insertion, plasma membrane targeting, and subunit-subunit interactions to be determined. In order to facilitate the design and interpretation o the mutagenesis experiments, single crystals both of intact subunits of Na,K- ATPase, and of selected domains of these polypeptides, will be prepared for x-ray diffraction analysis. Collectively, the data obtained from the chemical modification, sitedirected mutagenesis, and x-ray diffraction experiments should permit the development of a high-resolution model for the hydrolysis of ATP by Na,K-ATPase. Lastly, an experiment using anti-sense mRNA is described which has the potential to determine whether the beta subunit of Na,K-ATPase is essential for either enzyme activity or assembly.
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