The primary aim of the work outlined in this proposal 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 sideness 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, and changes in the phosphoryl potential. 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. We also plan to study the binding characteristics of K and its surrogates, cardiac glycosides and nuclotides to intact membranes and membrane components in an effort to define more precisely the substructure and the molecular events associated with membrane transport. In addition, we will continue our studies concerned with identifying the membrane process associated with Ca-induced K transport of human red cells by monitoring in resealed ghosts the Ca levels with entrapped arsenaoz III, membrane potentials and K permeability; in addition, we plan to label the membrane sites to determine their number and possibly to help in their isolation and subsequent characterization.
