The role of Na,K-ATPase alpha subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties of the Na,K-pump.

The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K-ATPase alpha subunit, in determining the voltage and extracellular K+ (K+(o)) dependence of enzyme-mediated ion transport, were examined in this study. HeLa cells expressing the alpha1 subunit of sheep Na,K-ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+ (37 degrees C). Na,K-pump current produced by the ouabain-resistant control enzyme (RD), containing amino acid substitutions Gln111Arg and Asn122Asp, displayed a membrane potential and K+(o) dependence similar to wild-type Na,K-ATPase during superfusion with 0 and 148 mM Na+-containing salt solutions. Additional substitution of alanine at Ser775 or Glu779 produced 155- and 15-fold increases, respectively, in the K+(o) concentration that half-maximally activated Na,K-pump current at 0 mV in extracellular Na+-free solutions. However, the voltage dependence of Na,K-pump current was unchanged in RD and alanine-substituted enzymes. Thus, large changes in apparent K+(o) affinity could be produced by mutations in the fifth transmembrane segment of the Na,K-ATPase with little effect on voltage-dependent properties of K+ transport. One interpretation of these results is that protein structures responsible for the kinetics of K+(o) binding and/or occlusion may be distinct, at least in part, from those that are responsible for the voltage dependence of K+(o) binding to the Na,K-ATPase.

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-ATPase and electrogenic Na+-Na+ exchange activity in 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 (K0(0.5)) of 13.4 +/- 0.6 mM and a low-affinity, high-capacity component with a K0(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.

Electrogenic K+ transport by the Na(+)-K+ pump in rat cardiac ventricular myocytes.

1. The involvement of electrogenic reaction steps in K+ transport by the Na+, K(+)-ATPase was determined in rat cardiac ventricular myocytes using whole-cell patch clamp techniques. 2. Under K(+)-K+ exchange conditions and in the presence of extracellular K+ or Tl+ at concentrations that stimulated submaximal levels of steady-state Na+,K(+)-ATPase activity, ouabain-sensitive transient currents were observed during ('on') and after ('off') step changes in membrane potential (Vm). 3. The quantity of charge moved during the transient currents depended, in a saturable manner, on the magnitude of the voltage step. Maximal ouabain-sensitive 'on' and 'off' charges were calculated to be 9.6 +/- 0.9 and 9.1 +/- 0.4 fC pF-1 (n = 4), respectively, with an effective valeney of 0.48 +/- 0.07 (n = 7). 4. Kinetics of the transient currents were independent of Vm and Tl+o at positive potentials, but became more rapid at increasingly negative Vm values in an ion concentration-dependent fashion. 5. Those data demonstrate that electrogenic steps participate in K+ transport by the Na+,K(+)-ATPase and that the electrogenic step is extracellular ion binding. 6. The temperature- and Vm-dependent properties of transient charge movements were compared under K(+) -K+ and Na(+) -Na+ exchange conditions. The data suggest that extracellular K+ and Na+ binding occur at different sites in the enzyme or to different enzyme conformations. The sum of the effective valencies, 1.14 +/- 0.12, demonstrates that the electrogenicity of extra-cellular ion binding can explain the Vm dependence of ion transport by the Na+,K(+)-ATPase.

Substitution of glutamic 779 with alanine in the Na,K-ATPase alpha subunit removes voltage dependence of ion transport.

The effects of changing Glu-779, located in the fifth transmembrane segment of the Na,K-ATPase alpha subunit, on the phosphorylation characteristics and ion transport properties of the enzyme were investigated. HeLa cells were transfected with cDNA coding the E779A substitution in an ouabain-resistant sheep alpha1 subunit (RD). Steady state phosphorylation stimulated by Na+ concentrations less than 20 mM or by imidazole were similar for RD and E779A enzymes, an indication that phosphorylation and Na+ occlusion were not altered by this mutation. With E779A enzyme, higher Na+ concentrations reduced the level of phosphoenzyme and stimulated Na-ATPase activity in the absence of K+. These effects were a consequence of Na+ increasing the rate of protein dephosphorylation. In voltage-clamped HeLa cells expressing E779A enzyme, a prominent electrogenic Na+-Na+ exchange was observed in the absence of extracellular K+. Thus, increased Na-ATPase activity and Na+-dependent dephosphorylation result from Na+ acting as a K+ congener with low affinity at extracellular binding sites. These data suggest that E779A does not directly participate in ion binding but does affect the connection between extracellular ion binding and intracellular enzyme dephosphorylation. In cells expressing control RD enzyme, Na,K-pump current was dependent on membrane potential and extracellular K+ concentration. However, Na,K-pump current in cells expressing E779A enzyme was voltage independent at all extracellular K+ tested. These results indicate that Glu-779 may be part of the access channel determining the voltage dependence of ion transport by the Na, K-ATPase.

Low affinity acceleration of the phosphorylation reaction of the Na,K-ATPase by ATP.

The maximum rate of phosphorylation (rm) of a highly purified Na,K-ATPase from red outer medulla of pig kidney was measured at 25 degrees C as a function of ATP concentration in media with Mg2+, Na+, and no K+. When rm was plotted as a function of the concentration of ATP a biphasic response was observed with a hyperbolic component of high affinity (Km = 15.7 +/- 2.6 microM) and low velocity ((rm)max = 460 +/- 40 nmol of Pi/(mg of protein.s)) plus a parabolic component which showed no saturation up to 1000 microM ATP, concentration at which rm was 1768.1 +/- 429.6 nmol Pi/(mg protein.s) (mean +/- S.E.; n = 3). This low affinity effect of ATP on the rate of phosphorylation disappeared when the Na,K-ATPase underwent turnover in medium without K+ suggesting that, like superphosphorylation (Peluffo, R. D., Garrahan, P. J., and Rega, A. F. (1992) J. Biol. Chem. 267, 6596-6601), it required the enzyme to be at rest. This property of the Na,K-ATPase was not predicted by the Albers-Post reaction scheme. The observed behavior of the enzyme could be simulated by a scheme that involves a resting enzyme (Er) functionally different from E1 or E2, which is able to bind three molecules of ATP, one with high and two with low affinity, and that after phosphorylation is converted into the phosphointermediates that are generally considered to participate in the reaction cycle described by Albers and Post.

Low affinity superphosphorylation of the Na,K-ATPase by ATP.

Pre-steady-state phosphorylation of purified Na,K-ATPase from red outer medulla of pig kidney was studied at 25 degrees C and an ample range of [tau-32P]ATP concentrations. At 10 microM ATP phosphorylation followed simple exponential kinetics reaching after 40 ms a steady level of 0.76 +/- 0.04 nmol of P/mg of protein with kapp = 73.0 +/- 6.5 s-1. At 500 microM ATP the time course of phosphorylation changed drastically, since the phosphoenzyme reached a level two to four times higher at a much higher rate (kapp greater than or equal to 370 s-1) and in about 40 ms dropped to the same steady level as with 10 microM ATP. This superphosphorylation was not observed in Na,K-ATPase undergoing turnover in a medium with Mg2+, Na+, and ATP, suggesting that it required the enzyme to be at rest. Superphosphorylation depended on Mg2+ and Na+ and was fully inhibited by ouabain and FITC. After denaturation the phosphoenzyme made by superphosphorylation had the electrophoretic mobility of the alpha-subunit of the Na,K-ATPase, and its hydrolysis was accelerated by hydroxylamine. On a molar basis, the stoichiometry of phosphate per ouabain bound was 2.40 +/- 0.60 after phosphorylation with 1000 microM ATP. The results are consistent with the idea that under proper conditions every functional Na,K-ATPase unit can accept two, or more, phosphates of rapid turnover from ATP.