The effect of holding potential on charge translocation by the Na+/K +-ATPase in the absence of potassium.

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 ADPi, 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.