Electrogenic Sodium–Sodium Exchange Carried Out by Na,k -Atpase Containing the Amino Acid Substitution Glu779ala

Abstract

Na,K -ATPase containing the amino acid substitution glutamate to alanine at position 779 of the α subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+–Na+ exchange activityin the absence of K +. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 ± 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K +. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K +. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala 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 concentrations tested (3–148 mM), exchange current was maximal at negative membrane potentials (VM), but decreased as VM became more positive. Analyzing this current at each VM with a Hill equation showed that Na+–Na+ exchange had a high-affinity, low-capacity component with an apparent Na+o affinity at 0 mV (K 00.5) of 13.4 ± 0.6 mM and a low-affinity, high-capacity component with a K 00.5 of 120 ± 13 mM (n = 17). Both high- and low-affinity exchange components were VM dependent, dissipating 30 ± 3% and 82 ± 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component 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 + congener; 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 the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47–59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K -ATPase.