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 (I_H), which may be exacerbated in NKA-subunit mutations associated with human disease. To elucidate the mechanisms of I_H, we studied the effects of intracellular ligands (transported ions, nucleotides, and beryllium fluoride) on I_H and, for comparison, on transient currents measured at normal Na⁺_o (Q_Na). Utilizing inside-out patches from Xenopus oocytes heterologously expressing NKA, we observed that 1) in the presence of Na⁺_i, I_H and Q_Na were both activated by ATP, but not ADP; 2) the [Na⁺]_i dependence of I_H in saturating ATP showed K_0.5,Na = 1.8 ± 0.2 mM and the [ATP] dependence at saturating [Na⁺]_i yielded K_0.5,ATP = 48 ± 11 μM (in comparison, Na⁺_i-dependent Q_Na yields K_0.5,Na = 0.8 ± 0.2 mM and K_0.5,ATP = 0.43 ± 0.03 μM; 3) ATP activated I_H in the presence of K⁺_i (∼15% of the I_H observed in Na⁺_i) only when Mg²⁺_i was also present; and 4) beryllium fluoride induced maximal I_H even in the absence of nucleotide. These data indicate that I_H 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 P_i (at the micromolar levels found in millimolar ATP solutions) promotes external release of occluded K⁺ by backdoor NKA phosphorylation. Maximal I_H through beryllium-fluorinated NKA indicates that this complex mimics ATP-bound E2P states.