Coupling the energy of ATP hydrolysis to the transport of sodium and potassium, the Na, K-ATPase of animal cell plasma membranes maintains the non-equilibrium ion distributions that are necessary for cellular function. The maintenance of high K and low Na levels inside the cell 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 this membrane transport process is vital to progress in all areas of human physiology, metabolism, and disease. While studies in the last twenty-five years have led to detailed knowledge of the steady-state characteristics of ion transport and of rapid changes in enzyme conformation we have only recently begun to assign the movements of Na and K to particular steps in the enzymatic cycle. 1) Our recent investigations have led to an understanding of the K-transporting phase of the cycle, involving an """"""""occluded"""""""" state of Na, K-ATPase in which K ions are hidden within the structure of the protein during their transport across the membrane. In the proposed work we will continue to use novel rapid kinetic techniques developed in this laboratory to study the events involved in Na and K transport, concentrating on events in the Na-transporting phase of the cycle. We will study the time course of Na release from Na K-ATPase during transport, and the formation and breakdown of a state in which Na is occluded. The Na translocation events will be temporally correlated with formation and breakdown of phosphorylated intermediates, with changes in conformational state of the protein, and with transmembrane charge movement. 2) We will also attempt to discover the identify of amino acids involved in cation transport, by labeling the Na, K-ATPase with photosensitive organic cations that become occluded at the Na or K sites. 3) Previous investigations have shown that in addition to the alpha and beta subunits of Na, K-ATPase, a low molecular weight membrane protein, a putative gamma subunit, is photolabeled by ouabain derivatives that are specifically bound to the enzyme. We propose to further characterize this protein with immunological and biochemical techniques, as well as through the isolation of cDNA clones encoding its peptide sequences. These molecular probes will be used to examine the level of expression in various tissues, to deduce the amino acid sequence of the peptide and to determine its functional role through in vitro expression. 4) Finally, we plan to use molecular biologic techniques to study the biosynthetic assembly, processing and sorting of Na, K-ATPase. We will transcribe mRNA from cDNA clones encoding the subunits of Na, K- ATPase, and inject this mRNA into Xenopus laevis oocytes. Immunofluorescence and immunoprecipitation will be used to determine the requirements for transport of individual or assembled subunits to the cell surface, as well as to determine the involvement of endogenous Xenopus components.
