Two‐Electrode Voltage‐Clamp Analysis of Na,K‐ATPase Asparagine 776 Mutants

Abstract

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 E(1)P(Na(+)) <--> E(2)P + 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(776) decreases the affinity of sodium at its extracellular binding site.

Displacement of the Na + /K + pump’s transmembrane domains demonstrates conserved conformational changes in P-type 2 ATPases

“…3A, dashed lines). Transient currents were slower at positive voltages and became faster at negative voltage due to the voltage-dependent rebinding of Na + , as it is observed in Na + /K + pumps of diverse origin (31)(32)(33)(34)(35)(36)(37). The fluorescence signal measured simultaneously in the same oocyte (Fig.…”