Effect of Na _i on activity and voltage dependence of the Na/K pump in adult rat cardiac myocytes

Abstract

We have measured the voltage dependence of the Na/K pump in isolated adult rat cardiac myocytes using the whole-cell patch-clamp technique. In the presence of 1–2 mM Ba and 0.1 mm Cd and nominally Ca-free, Na/K pump current (I _p) was measured as the change in current due to 1 mM ouabain. Voltage dependence of I _pwas measured between −140 and +40 or +60 mV using square voltage-pulse and voltage-ramp protocols, respectively. With 150 mM extracellular Na (Na _o ) and 5.4 mM extracellular K (K _o ), we found that the Na/K pump shows a strong positive voltage dependence between −140 and 0 mV and is voltage independent at positive potentials. Removing Na _o reduced the voltage dependence at negative potentials with no effect at positive potentials. When K _o was reduced, a negative slope appeared in the current-voltage (I-V) curve at positive potentials. We have investigated whether Na _i (intracellular Na) might also affect the voltage dependence of I _pby varying Na in the patch pipette (Na_pip) between 20 and 85 mM. We found, as expected, that I _pincreased markedly as Na_pip was raised, saturating at about 70 mM Na_pip under these conditions. In contast, while I _psaturated near +20 mV and declined to about 40% of maximum at −120 mV, there was no effect of Na_pip under these conditions. In contrast, while I _psaturated near +20 mV and declined to about 40% of maximum at −120 mV, there was no effect of Na_pip on the voltage dependence of I _p. This suggests that neither Na _i binding to the Na/K pump nor the conformational changes dependent on Na _i binding are voltage dependent. These results are consistent with extracellular ion binding within the field of the membrane but do not rule out the possibility that other steps, such as Na translocation, are also voltage dependent.