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Na(+)/K(+)-ATPase (NKA) exports 3Na(+) and imports 2K(+) at the expense of the hydrolysis of 1ATP under physiological conditions. In the absence of K(+), it can mediate electroneutral Na(+)/Na(+) exchange. In the electroneutral Na(+)/Na(+) exchange mode, NKA produces a transient current containing fast, medium and slow components in response to a sudden voltage step. These three components of the transient current demonstrate the sequential release of Na(+) ions from three binding sites. Our data from oocytes provide further experimental support for the existence of these components. Oligomycin is an NKA inhibitor that favors the 2Na(+)-occluded state without affecting the conformational state 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 blocked almost all of the K(+)-activated current, although the three components of the transient current showed different sensitivities to oligomycin. The oligomycin-inhibited charge movement measured using a P/4 protocol had a rate coefficient similar to the medium transient component. The fast component of the transient current elicited by a short voltage step also showed sensitivity to oligomycin. However, the slow component was not totally inhibited by oligomycin. Our results indicate that the second and third sodium ions might be released to the extracellular medium by a mechanism that is not shared by the first sodium ion. [ABSTRACT FROM AUTHOR]

The Na(+)/K(+)-ATPase exports 3Na(+) and imports 2K(+) at the expense of the hydrolysis of 1 ATP. In the absence of K(+), it carries on electroneutral, Na(+)-dependent transient charge movement (also known as "electroneutral Na(+)/Na(+) exchange mode") and produces a transient current containing faster and slower components in response to a sudden voltage step. Components with different speeds represent sequential release of Na(+) ions from three binding sites. The effect of holding potential on slow charge movement was studied in the presence of different concentrations of ADP(i), Na (i) (+) and Na (o) (+) with the intention of improving our understanding of Na (i) (+) binding. However, the manipulation of [ADP](i) and [Na(+)](i) did not cause as pronounced changes as predicted in the magnitude of charge movement (Q (tot)), which indicated that our experimental conditions were not able to backwardly drive reaction across the energy barrier to Na (i) (+) release/rebinding steps. On the contrary, lowering [Na(+)](o) caused evident dependence of Q (tot) on holding potential, with characteristics suggesting that pumps were escaping from E2P through the uncoupled Na(+) efflux activity. [ABSTRACT FROM AUTHOR]