Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala

Na,K-ATPase containing the amino acid substitution glutamate to alanine at position 779 of the alpha subunit (Glu779Ala) supports a high level of Na-A TPase and electrogenic Na+-Na+ exchange activity in the absence of K+. In m icrosomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 +/- 14 mM (n = 3). Further more, enzyme activity with 800 mM Na+ was found to be similar in the presen ce and absence of 20 mM K+. These results showed that Na+, with low affinit y, could stimulate enzyme turnover as effectively as K+. To gain further in sight into the mechanism of this enzyme activity, HeLa cells expressing Glu 779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K+-free solutions. Electrogenic Na+-Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na-o(+)) concentration. At all Na-o(+) c oncentrations tested (3-148 mM), exchange current was maximal at negative m embrane potentials (V-M), but decreased as V-M became more positive. Analyz ing this current at each V-M with a Hill equation showed that Na+-Na+ excha nge had a high-affinity, low-capacity component with a K-0.5(0) of 120 +/- 12 mM (n = 17). Both high- and low-affinity exchange components were V-M de pendent, dissipating 30 +/- 3% and 82 +/- 6% (n = 17) of the membrane diele ctric, respectively. The low-affinity, but not the high-affinity exchange c omponent was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+- Na+ exchange could be explained by Na-o(+) acting as a low-affinity K+ cong ener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na-o(+) binding at a Na+- specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K-ATPase was developed to simulate these data and the results of th e accompanying paper (Peluffo, R.D., J.M. Arguello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47-59). This model showed that alterations in the kine tics of extracellular ion-dependent reactions alone could explain the effec ts of Glu779Ala substitution on the Na,K-ATPase.