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 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.