The electrogenic sodium pump in guinea‐pig ventricular muscle: inhibition of pump current by cardiac glycosides

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Abstract
The inhibition of the electrogenic sodium pump in guinea-pig ventricular muscle by cardiac glycosides was studied with a voltage-clamp technique. Superfusion of the preparation with dihydro-ouabain (DHO) produced a reversible depolarization of up to 7 mV. When the membrane potential was clamped to a constant value near the resting potential, application of DHO produced a corresponding current change in the inward direction which reached a steady state in < 1 min. The drug-induced current change (ID) was the result of a parallel shift of the current-voltage relation. The contributions of a change in extracellular K or intracellular Na to the measured ID were very small. ID represents the blockage of the electrogenic pump current by DHO and that it is proportional to the number of drug molecules bound to the Na-K-ATPase in the intact cell. The dependence of ID on the concentration of DHO applied (5 .times. 10-6-8 .times. 10-4 M) was consistent with the predictions of the law of mass action for reversible 1- to 1 binding of the drug to the Na-K pump under equilibrium conditions. From a Scatchard-type plot, KD of DHO was determined to be 4.6 (.+-. 2.3) .times. 10-5 M. The steady-state pump current in the resting preparation was 0.81 .+-. 0.26 .mu.A/cm2. It contributed 6.4 .+-. 0.9 mV to the resting potential in Tyrode solution containing 3 mM-K. In the smallest preparations used, the measured time course of the onset and decay of ID agreed with the chemical kinetics of binding and unbinding calculated for various DHO concentrations. The rate constant of unbinding (k2) was 3.4 (.+-. 0.7) .times. 10-2 s-1 and the average rate constant of binding (k1) was 7.4 .times. 102 M-1 s-1. By comparing the effects of ouabain and DHO in the same preparation, the following estimates of the chemical constants of ouabain binding to the Na-K pump were obtained: KD .simeq. 1.5 .times. 10-6 M; k1 .simeq. 4 .times. 103 M-1 s-1; k2 .simeq. 6 .times. 10-3 s-1. An analysis of the transmembrane movements of Na and K in the steady state showed that the measured pump current density is consistent with a countertransport of 3 Na and 2 K ions.