THE KINETICS OF THE INHIBITION BY DIHYDROOUABAIN OF THE SODIUM-PUMP CURRENT IN SINGLE-RABBIT CARDIAC PURKINJE-CELLS

The kinetics of the inhibition by dihydroouabain (DHO) of the Na pump current were studied in isolated rabbit cardiac Purkinje cells by means of whole-cell recording. A fast exchange of the extracellular solution at the membrane of the cell studied was performed via two multibarrelled pipettes nearby. Judging from the steady-state inhibition of the Na pump current at various DHO concentrations half maximal inhibition occurred at 1 X 10(-5) mol/l DHO in a medium containing 2 mmol/l K+ and at 3.5 x 10(-5) mol/l DHO in a solution containing 10.8 mmol/l K+. Assuming a reversible one-to-one binding reaction, the time course of DHO binding and unbinding was analysed under a variety of conditions. It was shown that the antagonistic effect of K+ on the inhibition of the pump current was entirely due to a decrease of the association rate constant of DHO binding to the sodium pump at higher K+ concentrations. The association rate constant amounted to 1 X 10(4) (mol/l)-1 .s-1 at 2 mmol/l K+ and to 2.8 x 10(3) (mol/l)-1.-s-1 at 10.8 mmol/l K+. The dissociation rate constant of DHO unbinding remained unchanged (approximately 0.06 s-1). The equilibrium dissociation constant K(D) for the inhibition of the pump current by DHO decreased by a factor 3 to 5 if the Na+ concentration of the patch pipette solution was augmented from 5 to 50 mmol/l. Increasing DHO concentrations inhibited the pump current increasingly faster and to a larger extent. The K(D) values derived showed little variation with the concentration of DHO applied. It is concluded that the inhibition by DHO of the pump current in cardiac Purkinje cells can be adequately described by an one-to-one binding reaction between the cardioactive steroid and the pump molecules. The reaction is modulated by external K and internal Na ions.