Voltage dependence of the Na-K ATPase: measurements of ouabain-dependent membrane current and ouabain binding in oocytes of Xenopus laevis.
Ouabain- or dihydroouabain(DHO)-sensitive membrane currents and binding of 3H-ouabain were investigated under voltage-clamp conditions in full-grown prophase-arrested oocytes of Xenopus laevis. (1) The ouabain-sensitive current is outwardly directed and usually exhibits a maximum at about +20 mV. The occurrence of the maximum is not affected by application of blockers for passive K+ currents. In the presence of Ba2+ as a K+ channel blocker, the KI value for inhibition of the Na-K ATPase by ouabain is reduced by an order of magnitude, the number of binding sites is not affected. In K+-free solution (which inhibits the normal reaction cycle of the Na-K ATPase), addition of DHO has no significant effect on the remaining currents. (2) The voltage-dependence of the ouabain-sensitive current can be modulated. Reduction of extracellular Na+ increases the pump current at the resting potential and reduces the positive slope of the I-V curve. Simultaneously, the number of binding sites for ouabain is reduced by about 25%. Seasonal variations of an unknown factor affect the negative slope. (3) Modulation of the voltage-clamp conditions has no effect on the number of binding sites for ouabain, on current inhibition by ouabain, or on ouabain binding at different concentrations of the inhibitor. It is concluded that the ouabain-sensitive current is not significantly affected by passive permeabilities and that its current-voltage dependence reflects the voltage dependence of current generated by the Na-K pump. Since ouabain binding, also at non-maximal binding concentrations, is not affected by membrane potential, steps that affect the probability of the E2P state should be voltage-insensitive.
Schweigert
B
B
Max-Planck-Institut für Biophysik, Frankfurt/M, Federal Republic of Germany.
Lafaire
A V
AV
Schwarz
W
W
D000818:Animals; D001665:Binding Sites; D002463:Cell Membrane Permeability / Q000187:drug effects; D005260:Female; D066298:In Vitro Techniques; D008564:Membrane Potentials; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D010042:Ouabain / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014982:Xenopus laevis
D015221:Potassium Channels; D015222:Sodium Channels; C000977:dihydroouabain; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase