Voltage Dependent Reactions in the Non-Gastric H/K-ATPase

Abstract

The non-gastric H/K-ATPase (ngHKA) is the closest relative to the Na/K-ATPase (NKA). Its catalytic subunit associates with the NKA β1 subunit to form a functional, ouabain-sensitive HKA. To study ion binding and unbinding reactions in ngHKA, we measured 86Rb uptake and ouabain-sensitive (20 mM) currents under two-electrode voltage clamp in Na+-loaded Xenopus oocytes. Although HKA transports ions in an electroneutral fashion, HKA injected oocytes presented clamp speed-limited ouabain-sensitive charge movement in response to pulses between −180 to +40 mV from −50 mV in 125 mM external N-methyl D-glucamine (NMG). The charge-voltage curve did not saturate at extreme voltages at pH 7.6, but saturated at negative voltages at pH 5.0. The charge moved by a pulse from −50 to −180 mV was 1.29 ± 0.15 nC at pH 7.6 and 0.35 ± 0.08 nC at pH 5.0. This charge was absent in uninjected oocytes (60 ± 23 pC). External K+ abolished charge movement with mildly voltage-dependent K0.5 (∼ 0.15 mM at −180 mV), while the charge moved in the presence of Na+ at pH 7.6 presented nearly identical saturation at negative voltages as NMG at pH 5.0. This demonstrates the existence of mildly voltage-dependent transitions in ngHKAs which, like the charge movement in NKA, are inhibited by the external presence of the imported ion (K+) and modified by high concentrations of the exported ions (Na+ or H+, respectively). To further address the ion binding reactions and their selectivity, we mutated K793A in the catalytic subunit to make the pump electrogenic. As expected, this mutant concomitantly displayed 86Rb uptake and Rb+-induced steady-state outward currents as well as altered transient charge movement. Unexpectedly, K793A largely reduced the pump's affinity for K+o. Supported by NSF-MCB-1243842 & NIH-NS081570-01, to PA; GM 061583 to CG