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

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 (Ip) was measured as the change in current due to 1 mM ouabain. Voltage dependence of Ip was measured between -140 and +40 or +60 mV using square voltage-pulse and voltage-ramp protocols, respectively. With 150 mM extracellular Na (Nao) and 5.4 mM extracellular K (Ko), 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 Nao reduced the voltage dependence at negative potentials with no effect at positive potentials. When Ko was reduced, a negative slope appeared in the current-voltage (I-V) curve at positive potentials. We have investigated whether Nai (intracellular Na) might also affect the voltage dependence of Ip by varying Na in the patch pipette (Napip) between 20 and 85 mM. We found, as expected, that Ip increased markedly as Napip was raised, saturating at about 70 mM Napip under these conditions. In contrast, while Ip saturated near +20 mV and declined to about 40% of maximum at -120 mV, there was no effect of Napip under these conditions. In contrast, while Ip saturated near +20 mV and declined to about 40% of maximum at -120 mV, there was no effect of Napip on the voltage dependence of Ip. This suggests that neither Nai binding to the Na/K pump nor the conformational changes dependent on Nai 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.