Voltage dependence of Na translocation by the Na/K pump

Abstract

During each complete reaction cycle, the Na/K pump transports three Na ions out across the cell membrane and two K ions in. The resulting net extrusion of positive charge generates outward membrane current but, until now, it was unclear how that net charge movement occurs. Reasonable possibilities included a single positive charge moving outwards during Na translocation; or a single negative charge moving inwards during K translocation; or either positive or negative charges moving during both translocation steps, but in unequal quantities. Any step that involves net charge movement through the membrane must have voltage-dependent transition rates. Here we report measurements of transient, voltage-dependent, displacement currents generated by the pump when its normal Na/K transport cycle has been interrupted by removal of external K and it is thus constrained to carry out Na/Na exchange. The quantity and voltage sensitivity of the charge moved during these transient currents suggests that Na translocation includes a voltage-dependent transition involving movement of one positive charge across the membrane. This single step can thus fully account for the electrogenic nature of Na/K exchange. The result provides important new insight into the molecular mechanism of active cation transport.

Conformational dynamics of the Na + /K + -ATPase probed by voltage clamp fluorometry

“…There is general agreement, that Na ϩ -dependent transport steps are electrogenic ( Fig. 1 A, underlaid in gray) and kinetically coupled to the E 1 P-E 2 P conformational change (16)(17)(18)(19)(20)(21)(22)(23).…”

“…To allow for a more specific attribution of fluorescence signals to partial reactions of the Albers-Post cycle, we tested whether the fluorescence changes reflect the E 1 P-E 2 P transition under certain conditions, and performed voltage pulse experiments in the absence of K ϩ . Under these conditions, the Na ϩ ͞K ϩ -ATPase is restricted to Na ϩ ͞Na ϩ exchange (21). Because dephosphorylation in the absence of K ϩ is slow, the enzyme shuttles in a voltage-dependent manner almost exclusively between E 1 P and E 2 P, which allows the isolated investigation of the E 1 P-E 2 P conformational change.…”

“…Ouabain binds to the Na ϩ ͞K ϩ -ATPase in the E 2 P conformation (35,36), in agreement with the observed low fluorescence level (indicative of E 2 P). Transient currents in the Na ϩ ͞Na ϩ exchange mode are often defined as ouabain-sensitive currents in the literature (19,(21)(22)(23). The fluorescence data show that ouabain blocks the E 2 P-E 1 P transition, which also precludes transient charge translocation.…”

“…When plotted against the membrane potential, the amount of charge moved during a transient current follows a Boltzmann distribution (21), which is shown in Fig. 3D (Q-V curve, filled circles).…”

“…The main electrogenic events, namely sodium binding steps, occur on a microsecond time scale, but the detected transient charge movement is rate-limited by the conformational change E 2 P(Na) 7 E 1 P(Na) (19). Relaxation into a new distribution of states occurs with the sum of forward and backward reaction rate constants, and transient currents decay with first order kinetics (18)(19)(20)(21)(22)(23).…”