Three distinct and sequential steps in the release of sodium ions by the Na+/K+-ATPase

The Na+/K+ pump, a P-type ion-motive ATPase, exports three sodium ions and then imports two potassium ions in each transport cycle. Ions on one side o f the membrane bind to sites within the protein and become temporarily occl uded (trapped within the protein) before being released to the other side(1 ,2), but details of these occlusion and de-occlusion transitions remain obs cure for all P-type ATPases, If it is deprived of potassium ions, the Na+/K + pump is restricted to sodium translocation steps(3), at least one involvi ng charge movement through the membrane's electric field(4,5). Changes in m embrane potential alter the rate of such electrogenic reactions and so shif t the distribution of enzyme conformations. Here we use high-speed voltage jumps to initiate this redistribution and show that the resulting pre-stead y-state charge movements relax in three identifiable phases, apparently ref lecting de-occlusion and release of the three sodium ions. Reciprocal relat ionships among the sizes of these three charge components show that the thr ee sodium ions are de-occluded and released to the extracellular solution o ne at a time, in a strict order.