The Na+/K+ ATPase (NKA) exports 3Na+ and imports 2K+ at the expense of the hydrolysis of 1 ATP. In the absence of K+, it carries on electroneutral Na+/Na+ exchange and produces a transient current containing faster and slower components in response to a sudden voltage step. Components with different speeds, representing sequential release of Na+ ions from three binding sites, were demonstrated by analysis using exponential fit of experimental data. Our data provides experimental support that the slow component can be present independently. Oligomycin is a NKA inhibitor favoring the 2Na+-occluded state without affecting the conformational change of the NKA. We studied the effects of oligomycin on both K+ activated currents and transient currents in wild-type Bufo NKA and a mutant form of Bufo NKA, NKA: G813A. Oligomycin almost completely blocked K+ activated current and the binding of oligomycin was voltage independent. Inhibition affected faster components without affecting the slow component carried by the first Na+ release/rebinding. The inhibited component shown using the P/4 leakage subtraction protocol had a rate coefficient and voltage dependence of charge similar to the medium component. Our study indicates that Na+ is released to the extracellular medium through at least two pathways.
In another set of studies, the effect of holding potential on slow charge movement was studied in the presence of different concentrations of ADPi, Na+i or Na+o. This was done to improve our understanding of Na+i binding. However, the manipulation of [ADPi] and [Na+i] did not cause as pronounced changes as predicted in magnitude of charge movement (Qtot), which indicated that our experimental conditions were not able to backwardly drive reaction across energy barrier to Na+i release/rebinding steps. On the contrary, lowering [Na+o] caused evident dependence of Qtot on holding potential with characteristics suggesting pumps were escaping from E2P through the uncoupled Na+ efflux activity.