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Original Article

^a Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103
^b Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609
^c Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267
Department of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, 185 S. Orange Avenue, Newark, NJ 07103.973-972-7950

berlinjr{at}umdnj.edu

Na,K -ATPase containing the amino acid substitution glutamate to alanine at position 779 of the 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 (V_M), but decreased as V_M became more positive. Analyzing this current at each V_M 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 ⁰_0.5) of 13.4 ± 0.6 mM and a low-affinity, high-capacity component with a K ⁰_0.5 of 120 ± 13 mM (n = 17). Both high- and low-affinity exchange components were V_M 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.

Key Words: Na,K -pump • Na⁺–Na⁺ exchange current • HeLa cells • voltage clamp

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