Abstract
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 Mg(2+)(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.