Recently, it has been reported that actin stimulates purified Na+,K+- ATPase activity. This plan is aimed to characterize the role of actin and associated proteins on the efficiency and stoichiometry of ion transfer mediated by the enzyme cytoskeleton in renal epithelial cells. The dynamics of the modulation of Na-K pump by actin will also be studied at the cellular level. The function of the pump will be estimated not only by measuring ATP hydrolysis, but also 86Rb+ (K-like) uptake and Na+,K+ pump currents in intact cells. The effect of several factors that affect the state of aggregation of actin will be determined. Na,K-pump will be also characterized in a system that has a modified actin cytoskeleton: a melanoma cell strain which lacks the expression of actin-binding proteins. To further describe the molecular interaction between actin and the enzyme, experiments will be made in cells that express sodium pump molecules bearing punctual mutations in their putative actin-binding sites. These studies will contribute to elucidate the role of actin cytoskeleton on the regulation of the Na,K-pump in epithelial cells.
