Intracellular Requirements for Passive Proton Transport through the Na + ,K + -ATPase.

The Na+ ,K+ -ATPase (NKA or Na/K pump) hydrolyzes one ATP to exchange three intracellular Na+ (Na+ i ) for two extracellular K+ (K+ o ) across the plasma membrane by cycling through a set of reversible transitions between phosphorylated and dephosphorylated conformations, alternately opening ion-binding sites externally (E2) or internally (E1). With subsaturating [Na+ ]o and [K+ ]o , the phosphorylated E2P conformation passively imports protons generating an inward current (IH ), which may be exacerbated in NKA-subunit mutations associated with human disease. To elucidate the mechanisms of IH , we studied the effects of intracellular ligands (transported ions, nucleotides, and beryllium fluoride) on IH and, for comparison, on transient currents measured at normal Na+ o (QNa ). Utilizing inside-out patches from Xenopus oocytes heterologously expressing NKA, we observed that 1) in the presence of Na+ i , IH and QNa were both activated by ATP, but not ADP; 2) the [Na+ ]i dependence of IH in saturating ATP showed K0.5,Na  = 1.8 ± 0.2 mM and the [ATP] dependence at saturating [Na+ ]i yielded K0.5,ATP  = 48 ± 11 μM (in comparison, Na+ i -dependent QNa yields K0.5,Na  = 0.8 ± 0.2 mM and K0.5,ATP  = 0.43 ± 0.03 μM; 3) ATP activated IH in the presence of K+ i (∼15% of the IH observed in Na+ i ) only when Mg2+ i was also present; and 4) beryllium fluoride induced maximal IH  even in the absence of nucleotide. These data indicate that IH occurs when NKA is in an externally open E2P state with nucleotide bound, a conformation that can be reached through forward Na/K pump phosphorylation of E1, with Na+ i and ATP, or by backward binding of K+ i to E1, which drives the pump to the occluded E2(2K), where free Pi (at the micromolar levels found in millimolar ATP solutions) promotes external release of occluded K+ by backdoor NKA phosphorylation. Maximal IH through beryllium-fluorinated NKA indicates that this complex mimics ATP-bound E2P states.