The effect of the gamma modulator on Na/K pump activity of intact mammalian cells.

This study concerns the modulatory effects of the gamma modulator of the Na/K pump, in particular whether the effects seen in previous experiments with isolated membranes are relevant to Na/K pump behavior in intact mammalian cells. For this purpose, HeLa cells previously transfected with the rat Na/K catalytic subunit were used. The results show that both variants of the regulator, gammaa and gammab, decrease the apparent affinity of the pump for Na(+) and cause a modest increase in apparent ATP affinity as seen in measurements of ouabain-sensitive (86)Rb(K(+)) influx into cells in which ATP was varied using antimycin A and glucose. Equivalent results had been obtained previously in our analyses of Na,K-ATPase activity of membrane fragments, i.e., an increase in K(0.5(Na)) at high K(+) concentration and a decrease in K'(ATP). Comparison of clones of gamma-transfected and mock-transfected cells (with similar V(max) values) indicated that gamma causes a modest approximately 30% increase in the steady-state concentration of intracellular Na(+). Furthermore, for both gammaa and gammab, values of intracellular Na(+) were similar to those predicted from the kinetic constants, K(0.5(Na)) and V(max). Finally, there was a gamma-mediated increase in apparent affinity for extracellular K(+), which had not been detected in assays of permeabilized membranes.