The work outlined in this proposal is directed towards an understanding, on the molecular level, of the mechanism by which Na+ and K+ are actively moved across the plasma membranes of animal cells by the enzyme Na, K-ATPase, fueled by the breakdown of ATP. The maintenance of high K+ and low Na+ levels inside the cell by this process is of fundamental importance in control of cell volume, in the movement of other solutes and water across cell membranes, in the net movement of salt and water across epithelial cells, and in the electrical excitability of nerve and muscle. Thus an understanding of membrane transport is vital to progress in all areas of human physiology, metabolism, and disease. While studies in the last twenty years have led to detailed knowledge of the steady-state characteristics of ion transport and of rapid changes in enzyme conformation, it has not been possible to unambiguously assign the movements of Na+ and K+ to particular steps in the enzymatic cycle. In this work I will determine the time course of ion movements during a single turnover of the Na pump and relate them directly to enzyme conformational changes and phosphorylation of Na, K-ATPase. I have recently shown that membrane vesicles isolated from the outer medulla of mammalian kidney are very rich in Na, K-ATPase and constitute an excellent preparation for this study. Using a novel technique developed in this laboratory, the rapid kinetics of Na release from the extracellular face of the Na pump can be determined following a pulse of ATP delivered within the vesicles by photolysis of caged ATP; in other experiments the release of K+ from the inside face of the membrane will be examined, as will the time course of """"""""occlusion"""""""" of ions in the membrane interior. In a second approach, the electrical events associated with ion movements will be measured on the same time scale, both by using potential-sensitive dyes, and by applying the electrical patch-clamp technique to the vesicle membrane. Further studies will assess the effects of covalent modification of the Na, K-ATPase on the ion translocation events to gain insight into the relationship of structure to function. Results of this research will provide a basis for an understanding of the detailed mechanism by which the release of metabolic energy is coupled to transmembrane ion movements.
