Two-electrode voltage-clamp analysis of Na,K-ATPase asparagine 776 mutants

Steady-state and pre-steady-state currents of Asn(776) mutants of Na,K-ATPase are presented. The stationary current generated by N776Q strongly depends on the membrane potential, but has a negative slope, opposite to that of the wild-type enzyme. The apparent rate constant of the reaction sequence E1P(Na+) <----> E2P + Na+ of this mutant is rather independent of the membrane potential and is at resting and depolarizing membrane potential higher than that of the wild-type enzyme. Thus, the voltage-dependent increase of the rate coefficient of the wild type that is associated with extracellular Na+ rebinding is almost absent in the N776Q mutant. These findings indicate that dislocating the carboxamide group of Asn(77)6 decreases the affinity of sodium at its extracellular binding site.