Abstract
1. The involvement of electrogenic reaction steps in K+ transport by the Na+, K(+)-ATPase was determined in rat cardiac ventricular myocytes using whole-cell patch clamp techniques. 2. Under K(+)-K+ exchange conditions and in the presence of extracellular K+ or Tl+ at concentrations that stimulated submaximal levels of steady-state Na+,K(+)-ATPase activity, ouabain-sensitive transient currents were observed during ('on') and after ('off') step changes in membrane potential (Vm). 3. The quantity of charge moved during the transient currents depended, in a saturable manner, on the magnitude of the voltage step. Maximal ouabain-sensitive 'on' and 'off' charges were calculated to be 9.6 +/- 0.9 and 9.1 +/- 0.4 fC pF-1 (n = 4), respectively, with an effective valeney of 0.48 +/- 0.07 (n = 7). 4. Kinetics of the transient currents were independent of Vm and Tl+o at positive potentials, but became more rapid at increasingly negative Vm values in an ion concentration-dependent fashion. 5. Those data demonstrate that electrogenic steps participate in K+ transport by the Na+,K(+)-ATPase and that the electrogenic step is extracellular ion binding. 6. The temperature- and Vm-dependent properties of transient charge movements were compared under K(+) -K+ and Na(+) -Na+ exchange conditions. The data suggest that extracellular K+ and Na+ binding occur at different sites in the enzyme or to different enzyme conformations. The sum of the effective valencies, 1.14 +/- 0.12, demonstrates that the electrogenicity of extra-cellular ion binding can explain the Vm dependence of ion transport by the Na+,K(+)-ATPase.