Effect of holding potential on charge translocation by the Na+‐K+ ATPase in its Na+‐Na+ exchange mode

The effect of holding potential ( V h ) on charge movement ( Q ) mediated by extracellular Na + release and rebinding was measured using the cut‐open oocyte voltage clamp technique in Xenopus oocytes expressing Bufo marinus Na + ‐K + ATPase α1 and β1 subunits (conditions:100mM Na + o , 10mM Na + i, 5mM ADP, 5mMATP). The following reaction scheme was used to interpret the results:Decreasing [Na + ] i reduced Q presumably due to accumulation of the enzyme in E1 states. Hyperpolarization was expected to decrease Q by its effect on K m . However, Q at V h = −100 mV increased 1.19 fold compared to Q at V h = −20 mV. Lowering [Na + ] o to 25mM gave Q −100 =0.52 Q −20 . Lowering [Na + ] i to 1mM gave Q −100 =1.23 Q −20 . In the absence of ADP and at saturating [Na + ] i Q −100 =0.97 Q −20 . The result indicates that two reaction steps are V h dependent and have opposite effects on Q . When the reaction scheme is shifted to the right by lowering [Na + ] o or excluding ADP, V h affects the slow conformational change E1P ↔ E2P. Hyperpolarization drives more pumps into E1P leaving fewer pumps available for Na + o release and rebinding. When the reaction scheme is shifted to the left by lowering [Na + ] i , we propose that V h affects the C terminal positive charged voltage sensor R1010 and R1011. Hyperpolarization shifts the reaction scheme rightward making more pumps available for Na + o release and rebinding. Research is supported by NIH grant NS‐022979.