Inhibition of the sodium pump in guinea-pig ventricular muscle by dihydro-ouabain: effects of external potassium and sodium.

The inhibition of the electrogenic pump current in quiescent guinea-pig ventricular muscle by dihydro-ouabain (DHO) was studied with the three-micro-electrode voltage-clamp technique described previously (Daut, 1982c). From dose-response curves of the drug-induced current change (ID) the equilibrium dissociation constant of the binding of DHO to the Na-K pump (KD) and the electrogenic pump current flowing in the steady state (Ip) were inferred (Daut & Rüdel, 1982b). The external K concentration ([K]o) was varied between 2 and 4.5 mM (substituted by Na). KD was found to increase with increasing [K]o. A plot of log KD versus log [K]o gave a straight line with a slope of about 1.5. The time constants of the onset (tau on) and decay (tau off) of ID are supposed to represent the chemical kinetics of binding and unbinding of the drug (Daut & Rüdel, 1981, 1982b). Tau on was found to be inversely related to [K]o whereas tau off was found to be independent of [K]o. Ip was found to be independent of [K]o. This was interpreted to indicate that in the steady state Ip is mainly determined by the passive influx of Na into the cell, which may be relatively insensitive to small changes in [K]o. The effects of [K]o on the drug-induced current change are consistent with competitive inhibition of the binding of DHO to the Na-K pump. It is suggested that K ions and cardiac glycosides compete for extracellular binding sites on the same conformation of the Na-K pump. The external Na concentration ([Na]o) was varied between 147 and 49 mM (substituted by choline or Tris). Reduction of [Na]o produced a proportional decrease of Ip. This may be a consequence of the accompanying reduction of passive Na influx and the resulting decrease in intracellular Na activity (alpha iNa). Reduction of [Na]o markedly increased KD. This effect may be mediated by competition between Na and K at the K-loading sites of the pump and/or by separate modulatory Na-binding sites. It is concluded that the well known effects of external Na and K on the positive inotropic action of cardiac glycosides can be fully accounted for by the marked changes in the apparent binding affinity of the drug reported here.