Our primary aim is to gain insight into the molecular mechanisms responsible for the membrane transport of electrolytes in red blood cells and the function these mechanisms have in the regulation of cell volume. The work is oriented toward establishing the relationship between phosphorylation of the Na:K pump complex, and the sidedness of action of various ligands involved, with the translocation of the transported ions. Studies will also be directed toward defining the energy relationships and coupling mechanisms between transport and cell metabolism. We plan to study the pump stoichiometry of Na to K as a function of cell Na concentration, especially in human red cells. These studies will be carried out measuring net Na and K movements and the pump's electrogenic contribution to the membrane potential estimated by means of a fluorescence dye technique. Since cell volume and metabolism (lactate production) are perturbed under many of these situations, these parameters will also be followed together with the possible involvement of membrane phosphoglycerate kinase and Na,K-ATPase defining a membrane pool of ATP. Functional consequences of the membrane pool will be evaluated together with an attempt to label the proteins comprising the pool. We propose to characterize further the efflux of 32P-inorg that occurs in conjunction with Na in Uncoupled Na efflux. We will also try to understand the relationship between Na/Na exchange and Na-ATPase activities that are known to take place in the Uncoupled mode. In addition, we will study further a New mode of Na efflux carried out by the pump that is promoted by ADP + P-inorg that is inhibited by ATP and in which P-inorg is transported together with Na. We also propose to study the effects of varying the membrane potential on the different partial reactions or modes of the Na/K pump trying to identify potential-sensitive steps in the pump's reaction cycle. Variations to be studied include alterations in the phosphoryl potential and different sets of the gradients of Na and K across the cell's membrane.
