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Free keywords:
ATPase, Na+/K+-; ATPase, H+/K-; Patch-clamp; Current-voltage relationship; Proton transport; (Xenopusoocyte)
Abstract:
The Na+/K+-ATPase of an ouabain-resistant mutant of Torpedo californica and of rat was expressed in Xenopus oocytes by microinjection of mRNA coding for the alpha- and the beta-subunit of the protein. Electrophysiological measurements were performed by means of the extracellular giant-patch clamp technique. The pump currents were analyzed in nominal absence of extracellular Na+ and K+ ions. Under these conditions strongly inward rectifying, ouabain-sensitive current was detected with reversal potentials depending on extracellular pH. Presence or absence of intracellular Na+ or K+ ions had nearly no effect on the inward currents, removal of intracellular ATP caused their inhibition. The reversal potentials of the currents generated by the rat pump was shifted by 82.7 ± 5.4 mV per 10-fold increase of extracellular proton concentration. This refers to an effective valency of 0.71 ± 0.05. In the absence of a transmembrane proton gradient the pump current reversed at -64.2 ± 4.4 mV. These findings are not compatible with a proton conducting channel formed by the pump molecule (Wang and Horisberger (1995) Am. J. Physiol. CP 37, C590-595). Therefore, a kinetic model based on the Post-Albers scheme with a modification derived from the reaction scheme of the gastric H+/K+-ATPase is proposed. Together with voltage-dependent binding of extracellular protons, this model is compatible with the observe pump currents.
