The proposed research will develop cell culture model systems to study the consequences of regulation of the sodium- and potassium-activated ATPase, the sodium pump. This enzyme is found in the plasma membrane of cells in all vertebrates and is evolutionarily related to cation transporting ATPases in lower eukaryotes and bacteria. Regulation of the ATPase will be modeled by generating gene amplified cell lines that have a range of expression of the sodium pump. Amplification of the genes for the sodium pump will be accomplished either by step-selection with ouabain or by transfection with minute chromosomes from C+, a HeLa cell line expressing unstable resistance to ouabain. The sodium pump is responsible for the generation of the sodium electrochemical potential across the plasma membrane, as well as the membrane potential. Thus, regulation of this enzyme may have profound consequences on the physiology of the organism. Evidence that such regulation may actually occur includes the remarkable evolutionary conservation of an inhibitory site on the enzyme which has been proposed to be the target of some endogenous natriuretic factors. Compounds which bind at this site include the important pharmacological probes for this activity, the alkaloids, digitalis and ouabain. This research should provide new information concerning the effect on cells of altered activity of the enzyme which "pumps" sodium out and potassium in, and which thus regulates the ionic composition of the cytoplasm, and contributes to the maintenance of the physiologically important, electrical potential across cell membranes. In addition, this research at an undergraduate institution will provide an opportunity for college students to participate in modern experiments in cell biology, and should stimulate their interest in research careers.
