Modulation of pump function by mutations in the first transmembrane region of Na(+)-K(+)-ATPase alpha 1-subunit.

The Cys in the first transmembrane region of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) alpha 1-subunit has been shown to be a critical determinant of cardiac glycoside binding. To study the role of this Cys on ion transport activity, we measured pump currents in HeLa cells expressing wild-type or mutant alpha 1-subunit cDNAs. The endogenous ouabainsensitive Na(+)-K(+)-ATPase was selectively inhibited by growing the cells in 0.1 microM ouabain. A Cys-to-Tyr substituted mutant exhibited decreased sensitivity to digitoxin but not digoxin compared with wild type. The decreased affinity for digitoxin was due to a faster dissociation rate. In contrast, the Cys-to-Ala substitution did not significantly alter the sensitivity to digitoxin or digoxin. Both wild-type and mutant cells displayed marked external K(+)-dependent pump currents; however, the affinity for K+ was reduced by the mutations. The decrease in K+ affinity was due to a slower association rate. The results show that the Cys that interacts with cardiac glycosides also participates in the sensitivity of the pump to external K+.