Na,K-ATPase, which is also called the sodium pump, is a membrane-embedded protein that is found in all animal cells. The Na,K-ATPase couples the hydrolysis of intracellular ATP to the transport of Na+ ions out of cells and K+ ions into cells, thereby participating in the mechanism of fluid and electrolyte balance in animals. The enzyme has been implicated in the etiology of essential hypertension, and as the only known receptor for cardiac glycosides such as digitalis and ouabain, is the target of pharmacologic intervention in congestive heart failure. Despite the important role of Na, K-ATPase in animal cell physiology, the mechanism of active transport catalyzed by this protein is unknown. As one approach to the resolution of this problem, this application describes several experiments designed to obtain structural data about Na,K-ATPase. The primary experimental tool to be used is molecular biology. Within this context, however, significant effort will also be placed on the use of physical methods for determination of the structure of the ATP binding site of the protein using preparations obtained from recombinant DNA technology. Among the specific aims of the project are the use of site-- directed mutagenesis, and chimeric molecules prepared from Na,K-ATPase and gastric H,K-ATPase, to identify amino acids that are located within the binding sites of Na, K-ATPase for ATP, cardiac glycosides, and the monovalent cations Na+ and K+. These mutant Na,K-ATPase molecules, the wild type Na, K-ATPase, and the chimeric pumps will be expressed in yeast cells, and measurements will be made on membranes or vesicles prepared from the yeast cells. Yeast cells have been chosen for this work because they do not contain endogenous Na,K-ATPase. The expression of Na,K-ATPase in yeast cells will also be used to determine whether the beta subunit has any function in Na,K-ATPase enzymatic activity apart from its known role in protein folding and maturation. The enzymatic characteristics of sodium pumps assembled from different isoforms of alpha and beta subunits will be measured in order to determine whether regulation of isoform expression and assembly may represent a form of regulation of Na,K-ATPase activity in cells. Isolated structural domains of Na,K-ATPase will be expressed in bacterial cells, and will be examined for the ability to bind ATP. Use of these preparations in NMR and diffraction experiments is described, with the goal of obtaining a high- resolution structure for this part of the Na,K-ATPase. Collectively, the data from the experiments described in this application should permit the development of a high-resolution model for the binding and hydrolysis of ATP by Na,K-ATPase, and will define the locations of other functionally significant sites within the protein structure.