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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

	ABSTRACT

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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

	Introduction

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After making the amino acid substitution glutamate to alanine at residue 779 of the subunit (Glu779Ala), Na,K -ATPase mediates an extracellular K ⁺ (K ⁺_o)-activated current whose amplitude is practically unchanged over a wide range of membrane potentials in the presence of high (148 mM) extracellular Na⁺. The inability of membrane potential (V_M) to affect current amplitude is observed at widely varying K ⁺_o concentrations, including those well below the concentration for half maximal current activation (Argüello et al. 1996). This behavior contrasts sharply with that of native enzyme, which shows a pronounced V_M dependence in transport rate under similar conditions (Gadsby et al. 1985; Gadsby and Nakao 1989; Bielen et al. 1991; Rakowski et al. 1991; Sagar and Rakowski 1994; Berlin and Peluffo 1997). Understanding the reason for these changes in the mutant enzyme is complicated by the presence of a high level of electrogenic Na⁺–Na⁺ exchange (Argüello et al. 1996) and Na-ATPase activity (Vilsen 1995; Argüello et al. 1996; Koster et al. 1996) that occurs in the absence of K ⁺_o.

Given the unexpected changes in current generated by Glu779Ala-containing enzyme, additional amino acid substitutions at residue 779 were investigated. One substitution, glutamate to glutamine (Glu779Gln), also resulted in K ⁺_o-activated currents mediated by the Na,K -ATPase that were V_M independent over a broad range of K ⁺_o and V_M in extracellular Na⁺ (Na⁺_o)-containing solutions. However, unlike Glu779Ala enzyme, electrogenic Na⁺–Na⁺ exchange was not measurable (Peluffo et al. 1997). These results showed that the V_M dependence of electrogenic Na⁺–K ⁺ exchange (i.e., Na,K -pump current) in the presence of Na⁺_o can be quite different for wild-type enzyme and variant enzymes containing substitutions at residue 779.

Membrane potential-dependent behavior of the Na,K -ATPase is due to the electrogenicity and kinetics of reactions controlling the ion transport cycle (Hansen et al. 1981). The preceding paper (Peluffo et al. 2000) showed that the electrogenicity and kinetics of K ⁺_o-dependent reactions are not dramatically affected in enzyme containing the substitution Glu779Gln. Thus, another explanation for the lack of V_M dependence for Na,K -pump current in Na⁺_o-containing solutions is that the electrogenicity and/or kinetics of Na⁺_o-dependent reactions are affected by Glu779Ala and Glu779Gln substitutions. In this regard, the high rate of electrogenic Na⁺–Na⁺ exchange activity by Glu779Ala enzyme becomes extremely fortuitous because Na⁺_o interactions with the Na,K -ATPase can be studied in the absence of K ⁺_o.

This study demonstrates that Na⁺_o-dependent reactions of Glu779Ala enzyme are highly V_M dependent and suggests that Na⁺_o activates enzyme turnover at K ⁺_o and Na⁺_o binding sites, similar to those in wild-type Na,K -ATPase. Thus, in addition to effects on K ⁺_o binding reactions (Peluffo et al. 2000), residue 779 may play a role in the kinetics of Na⁺_o binding and/or occlusion reactions.

Using data in this and previous studies (Argüello et al. 1996; Peluffo et al. 1997, Peluffo et al. 2000), a model is developed to explain the V_M and ion-dependent properties of electrogenic Na⁺–K ⁺ and Na⁺–Na⁺ exchange in Glu779Ala-containing enzyme. This model shows how alterations in the kinetics of ion binding reactions can explain the observed changes in V_M-dependent behavior of mutant enzymes.

Finally, the V_M-dependent properties of electrogenic Na⁺–Na⁺ exchange have not been studied, owing to the slow kinetics of this exchange mechanism in native enzyme. The present results, therefore, may also provide mechanistic information about electrogenic Na⁺–Na⁺ exchange in wild-type Na,K -ATPase.

	Methods

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Mutagenesis, transfection, and cell culture protocols were similar to the previous paper (Peluffo et al. 2000). Patch-clamp techniques were also similar. However, in some experiments, the intracellular electrode solution contained 11 mM ADP, as in "(Na⁺ salt)", 4 mM MgATP, and 9.2 mM MgCl₂, but omitted phosphocreatine and pyruvate. This solution was calculated to have a free ADP concentration of 2 mM (MaxChelator, supplied by Chris Patton, Hopkins Marine Station, Pacific Grove, CA). After establishing a whole-cell voltage clamp, the superfusion solution was switched to a K ⁺-free solution containing various concentrations of Na⁺. Na⁺ concentration in these solutions was changed by equimolar substitution of tetramethylammonium ion (TMA) with total monovalent cation concentration ([Na⁺] + [TMA]) equal to 148 mM. Electrogenic Na⁺–Na⁺ exchange mediated by heterologous enzyme was calculated as the ouabain-sensitive difference current recorded in the presence of 1 µM and 10 mM ouabain. Enzyme activity assays were performed as previously outlined (Argüello et al. 1996) except that total ionic strength was maintained at 1 M with choline chloride.

	Results

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Na-ATPase Activity
Na,K -ATPase enzyme containing the substitution Glu779Ala in the subunit has been reported to support Na-ATPase activity in the absence of K ⁺ that is 30–60% of maximal Na,K -ATPase activity, a level that is much higher than wild-type enzyme (Vilsen 1995; Argüello et al. 1996; Koster et al. 1996). However, these studies were conducted over a limited range of Na⁺ concentrations, up to 200 mM. To determine the Na⁺-dependent activation of this enzyme more completely, activity assays were performed at Na⁺ concentrations up to 800 mM (Fig. 1). Na,K -ATPase activity, measured in the presence of 20 mM K ⁺ and various concentrations of Na⁺, was activated by Na⁺ with a half maximal concentration (K _0.5) of 10.8 ± 2.9 mM (Hill coefficient equal to 1.0 ± 0.2), consistent with previous reports (Karlish and Stein 1985; Nakao and Gadsby 1989). At Na⁺ concentrations >200 mM, enzyme activity decreased slightly from a maximum, probably due to competition between Na⁺ and K ⁺ at K ⁺ binding sites in the enzyme (Skou 1960). In contrast, Na⁺-dependent activation of Na-ATPase activity showed a monotonic concentration dependence with a K _0.5 of 161 ± 14 mM (Hill coefficient fixed at 1). Maximal Na-ATPase activity was calculated to be 79 ± 3% of maximal Na,K -ATPase activity, but, at the highest Na⁺ concentrations, the levels of Na-ATPase and Na,K -ATPase activity were not significantly different. Thus, this mutant enzyme was stimulated by Na⁺ with a much lower apparent affinity than K ⁺, as has been previously reported (Argüello et al. 1996), and, at high enough concentrations, Na⁺-activated enzyme turnover rates were similar to those observed in the presence of K ⁺. These results suggest that Na⁺ can maximally activate enzyme activity, but at concentrations higher than K ⁺.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Na+ dependence of Na-ATPase activity. Enzyme activity corresponding to heterologous protein was calculated as the difference in activity measured with 1 µM and 10 mM ouabain. Total ionic strength was maintained at 1 M with choline chloride. Na,K -ATPase activity () was measured in the presence of 20 mM K Cl, and Na-ATPase activity was measured in K Cl-free solutions (•). The solid curves were fitted to the data using a Hill equation. The best-fit values for K 0.5 were 10.8 ± 2.9 mM (Hill coefficient, 1.0 ± 0.2) and 161 ± 14 mM (Hill coefficient fixed at 1) for Na,K -ATPase and Na-ATPase activity, respectively, determined in triplicate. The dotted curve was drawn by eye.

Current activation was measured with Na⁺_o concentrations ranging from 0 to 148 mM, while cells held at –40 mV were superfused in K ⁺_o-free solutions. Fig. 2 shows the effect of increasing ouabain concentration in the superfusion solution from 1 µM to 10 mM at the indicated Na⁺_o concentrations. Adding 10 mM ouabain produced an inward shift in holding current, the magnitude of which depended on Na⁺_o. The transient increase in current observed during the solution switch at intermediate Na⁺_o concentrations reflected an artifact due to the presence of 148 mM Na⁺ solution in the dead space of the superfusion bath. In Na⁺_o- and K ⁺_o-free solution, ouabain-sensitive changes in current were not observed (Fig. 2, bottom). This last result, in particular, shows that current arises from Na⁺–Na⁺ exchange rather than uncoupled Na⁺ efflux.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Extracellular Na+ dependence of ouabain-sensitive current. Cells were held at –40 mV during superfusion with solutions containing the Na+o concentration indicated to the left of each current tracing. Ouabain concentration in the superfusion solution was increased from 1 µM to 10 mM as indicated.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Current–voltage relationships. Membrane current measured in the presence of 1 µM (•) and 10 mM () ouabain during superfusion with solutions containing 12.5 (A) and 148 (B) mM Na+o.

Summary results for experiments at a variety of Na⁺_o concentrations are shown in Fig. 4 as ouabain-sensitive difference currents. Maximal Na⁺–Na⁺ exchange current at each Na⁺_o concentration was observed at negative V_M with current density decreasing as V_M became more positive. The voltage dependence of ouabain-sensitive difference current in the presence of Na⁺_o contrasts with the relative V_M independence of K ⁺_o-activated current observed in 148 mM Na⁺_o-containing solution (Argüello et al. 1996; Peluffo et al. 1997) and shows that some interactions of Na⁺_o with the enzyme involve V_M-dependent reactions.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Na+o and VM dependence of electrogenic Na+–Na+ exchange. Average ouabain-sensitive difference currents are plotted at the following Na+o concentrations (mM): 3.1 (), 9.4 (•), 50 (), 75 (), 100 (), and 148 (). Difference currents were calculated by subtracting current measured in the presence of 10 mM ouabain from current measured in 1 µM ouabain-containing solutions. The solid curves were fit to the data using equation A14 in Sagar and Rakowski 1994, modified for Na+–Na+ exchange in K +-free conditions. The number of cells voltage clamped at each Na+o was between 3 and 10.

Fig. 4 also shows a small positive slope in the I-V relationship with 9.4 mM Na⁺_o at potentials more negative than –80 mV. At several low Na⁺_o concentrations, such positive slopes were sometimes observed at the most negative potentials tested. Given their small size and the increased variability of current at these V_M, we did not investigate this point further.

To examine the V_M dependence of current more carefully, current density at selected V_M was plotted as a function of Na⁺_o concentration (Fig. 5). Viewed in this manner, it was immediately obvious that Na⁺_o dependence of current activation was not described by Michaelis-Menten kinetics. At the most negative V_M, current activation was a biphasic function of Na⁺_o. Even at positive V_M, current density showed a rapid increase below 25 mM Na⁺_o, along with an additional component of current activation that occurred at higher Na⁺_o concentrations. Given this complex dependence on Na⁺_o, ouabain-sensitive current density data at each V_M was fitted with a two-component Hill equation,

(1)

which included exchange current activated with relatively high (h) and low (l) affinities for Na⁺_o. Maximum overall current (i.e., I_max,h + I_max,l) was assumed to be constant. The solid curves in Fig. 5 are best-fit functions to the data at –100, 0, and +60 mV. In all cases, the Hill coefficient (_h) for the high affinity component of current was assumed to be equal to 1. This assumption was used because the limited number of low Na⁺_o concentrations tested would not allow an accurate estimate of the Hill coefficient, and since no sigmoidicity was apparent in the data, a value of 1 for _h seemed reasonable. was fitted to the data at all V_M. The resulting values of K _h and K _l, the Na⁺_o concentrations for half-maximal activation of the high and low affinity current components, respectively, are plotted separately in Fig. 6. Both K _h and K _l tended towards larger values as V_M became more positive, a result that suggests both current components are V_M dependent.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 5 VM dependence of Na+–Na+ exchange activation by Na+o. Ouabain-sensitive difference currents in Fig. 4 are replotted using data measured at –100 (•), 0 (), and +60 () mV. The solid curves are functions fit to the data using (see text for details). The values of Na+o concentration that produce half-maximal activation of the high Na+o affinity current component (K h) were calculated to be 3.8 ± 3.0, 13.4 ± 6.0, and 25.0 ± 2.5 mM at –100, 0, and +60 mV, respectively. The Hill coefficient (h) was fixed at a value of 1. The Na+o concentrations to produce half-maximal activation of the low Na+o affinity current component (K l) were calculated to be 75 ± 5, 120 ± 11, and 514 ± 88 mM, and the Hill coefficients (l) were calculated to be 3.0 ± 0.7, 2.4 ± 1.4, and 1.6 ± 0.7 at –100, 0, and +60 mV, respectively.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 6 VM dependence of the low and high Na+o affinity components of Na+–Na+ exchange. Ouabain-sensitive current at each VM was fitted with as in Fig. 5, and the resulting K h () and K l (•) are plotted versus VM. The solid curves were fitted to the data for K h and K l using and , respectively. The best-fit parameters for the high Na+o affinity current component were K h0 = 13.4 ± 0.6 mM and h = 0.30 ± 0.03 (n = 17). The best-fit parameters for the low Na+o affinity current component were K l0 = 120 ± 13 mM, l = 0.82 ± 0.06, and ii = 1.68 ± 0.07.

Given the apparent ability of Na⁺_o to act as a K ⁺_o-like congener, we first attempted to analyze the V_M-dependent properties of Na⁺–Na⁺ exchange current based on a pseudo two-state model similar to that used to analyze Na,K -pump current in the previous paper. Thus, by analogy to in Peluffo et al. 2000, the fraction of the membrane dielectric (_x) dissipated during Na⁺_o-dependent activation can be estimated by the following:

(2)

where x represents the high or low affinity component of exchange current, K _x⁰ is the concentration for half maximal current activation at 0 mV, and U is dimensionless V_M. As pointed out in Peluffo et al. 2000, this equation (and , below) does not imply a particular reaction mechanism for Na⁺–Na⁺ exchange.

Fitting to the data for the high affinity component of exchange current (Fig. 6, ) showed that K _h⁰ equaled 13.4 ± 0.6 mM and _h equaled 0.30 ± 0.03. Of particular note, _h is similar to the fraction of the membrane electric field dissipated by K ⁺_o during activation of Na,K -pump current mediated by Glu779Ala enzyme in Na⁺_o-free solutions.

We also attempted to fit to the K _l values shown in Fig. 6; however, this equation did not satisfactorily describe the data regardless of the weighting procedure (not shown). The inability to fit these data with a function in the form of again suggested that the activation of Na⁺–Na⁺ exchange by Na⁺_o was more complicated than a K ⁺_o congener-like action. For this reason, we attempted to fit the data for the low-affinity component of current with an equation analogous to in the preceding paper (Peluffo et al. 2000), in which we assume that Na⁺_o has two actions, one to activate current and the other to inhibit forward cycling of the enzyme (see DISCUSSION). Given these assumptions, the V_M dependence of K _l was analyzed by:

(3)

where K _l⁰ is K _l at 0 mV, B is the product of V_M-independent rate constants, and _ll and _ii are the products of the Hill coefficient and fractional distance for the low-affinity activating and inhibitory actions of Na⁺_o, respectively. Using the average value of _l determined above, was fitted to the data in Fig. 6. The K _l⁰ for Na⁺_o activation, 120 ± 13 mM (n = 17), was similar to the Na⁺ concentration for half-maximal activation of Na-ATPase activity (Fig. 1). This result is consistent with the suggestion that electrogenic Na⁺–Na⁺ exchange is the functional manifestation of Na-ATPase activity measured in vitro (Argüello et al. 1996).

As expected from the steep negative slope of the I-V relationships (Fig. 4), the low affinity reaction component dissipated over 80% of the membrane dielectric, _l = 0.82 ± 0.07 (n = 17). This high degree of electrogenicity is similar to that reported for Na⁺_o rebinding to wild-type Na,K -ATPase (Nakao and Gadsby 1986; Rakowski 1993; Heyse et al. 1994; Hilgemann 1994; Peluffo and Berlin 1997). These data show that low affinity activation of Na⁺–Na⁺ exchange occurs by a mechanism different than K ⁺_o-dependent activation of enzyme turnover and suggests again that Na⁺_o is not simply acting as a K ⁺ congener.

Relationship to Electroneutral Na⁺–Na⁺ Exchange
In the absence of K ⁺_o, wild-type Na,K -ATPase also carries out Na⁺–Na⁺ exchange that has one-to-one stoichiometry (Garrahan and Glynn 1967a; Abercrombie and De Weer 1978), is highly V_M-dependent (Rakowski et al. 1989; Gadsby et al. 1993), and has a strong dependence on ADP (De Weer 1970; Glynn and Hoffman 1971). Therefore, to determine the relationship between this electroneutral exchange and the electrogenic Na⁺–Na⁺ exchange measured with Glu779Ala enzyme, experiments compared the Na⁺_o and V_M dependence of Na⁺–Na⁺ exchange current with and without 2 mM free ADP in the patch electrode solution. Fig. 7 shows that, in the presence of 25 mM Na⁺_o, a concentration chosen primarily to stimulate the high affinity component of electrogenic exchange, increasing free ADP concentration in the patch electrode from 0 to 2 mM had little effect on the amplitude or V_M dependence of ouabain-sensitive current. In the presence of 148 mM Na⁺_o, increasing ADP concentration substantially reduced membrane current at all V_M. Even so, the steep negative slope in the I-V relationship remained, consistent with the presence of a highly V_M-dependent reaction in the remaining current. Thus, the high affinity component of electrogenic exchange appears to be ADP insensitive, while the low affinity exchange component is ADP sensitive.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Effect of ADP on Na+–Na+ exchange current. The VM dependence of ouabain-sensitive difference current is shown for cells voltage clamped with electrodes containing 0 () or 2 (•) mM free ADP. Experiments were performed while cells were superfused in solutions containing 25 (A) and 148 (B) mM Na+. The displayed data points are average values from three and four cells in 25 mM Na+o and from three and seven cells in 148 mM Na+o in the presence and absence of ADP, respectively.

	Discussion

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Na⁺–Na⁺ Exchange in Glu779Ala Enzyme
Na,K -ATPase containing Glu779Ala has the unique property that Na⁺ can stimulate enzyme activity in the absence of K ⁺ at rates much higher than with wild-type enzyme (Vilsen 1995; Argüello et al. 1996; Koster et al. 1996). The present results demonstrate that the rate of this Na-ATPase activity can be comparable with that of Na,K -ATPase activity, albeit at very high Na⁺ concentrations. Thus the principal question of this study is the mechanism by which Na⁺ promotes enzyme activity in this mutant enzyme.

Na⁺-stimulated enzyme activity is clearly electrogenic, since ouabain-sensitive currents are observed, and it does not represent uncoupled Na⁺ transport because Na⁺_o is required to activate current (Fig. 2; Argüello et al. 1996). Thus, Na-ATPase activity appears to be the in vitro manifestation of electrogenic Na⁺–Na⁺ exchange.

Na⁺_o-dependent activation of electrogenic Na⁺–Na⁺ exchange in Glu779Ala enzyme shows a biphasic dependence on Na⁺_o concentration (Fig. 5). Biphasic steady state reaction kinetics have previously been interpreted as indicative of (a) multiple enzyme isoforms catalyzing the same reaction, (b) complex allosteric substrate interactions in a multi-site enzyme (Segel 1975), or (c) kinetic heterogeneity in the reaction sequence of an enzyme (Froehlich and Fendler 1991). We can exclude the first possibility in our expression system; however, our data do not allow us to distinguish between the latter two possibilities. Nonetheless, kinetic heterogeneity has been previously postulated for the Na,K -ATPase (Froehlich and Fendler 1991; Post and Klodos 1996), and such a postulate simplifies data analysis by allowing us to separate Na⁺–Na⁺ exchange current into high and low Na⁺_o affinity components, as in . The high affinity component has a maximal velocity at –100 mV that is 24% of calculated maximal Na⁺–Na⁺ exchange current, and its apparent affinity is well described by an equation analogous to used to analyze Na,K -pump current in the previous paper (Peluffo et al. 2000). Using this model, our analysis suggested that Na⁺_o-dependent reactions dissipate 30% of the membrane electric field during Na⁺–Na⁺ exchange. This fractional distance for Na⁺_o is similar to that for K ⁺_o activation of Na,K -pump current (Sagar and Rakowski 1994; Berlin and Peluffo 1997).

The low-affinity component of Na⁺–Na⁺ exchange current is highly V_M-dependent and accounts for 76% of calculated maximal Na⁺–Na⁺ exchange current at –100 mV. Using , which is based on a pseudo three-state scheme for the Na,K -ATPase (Sagar and Rakowski 1994), we found that Na⁺_o-dependent activation dissipated 80% of the membrane electric field. It is interesting that the V_M dependence of this reaction appears to be quite similar to that for Na⁺_o rebinding in wild-type enzyme (Gadsby et al. 1993). Thus, it is tempting to postulate that activation of low-affinity electrogenic Na⁺–Na⁺ exchange in Glu779Ala enzyme occurs at a Na⁺-specific site.

Biphasic activation of Na-ATPase activity is not apparent in Fig. 1. However, the lowest Na⁺ concentration tested in these assays was still several-fold higher than the K ⁰_0.5 for Na⁺_o of the high affinity current component. The Na⁺ concentration for half-maximal activation of Na-ATPase activity is similar to that of the low Na⁺_o affinity current component. Thus, Fig. 1 probably shows activation of enzyme activity that is equivalent to the low affinity component of Na⁺–Na⁺ exchange.

Na⁺–Na⁺ Exchange Reactions by the Na,K -ATPase
Two types of Na⁺–Na⁺ exchange reactions have been previously reported in wild-type Na,K -ATPase (Glynn 1985). Electroneutral exchange is a one-to-one Na⁺ counter-transport mechanism (Garrahan and Glynn 1967a; Abercrombie and De Weer 1978) that involves no net hydrolysis of ATP (Garrahan and Glynn 1967b), is stimulated by ADP (De Weer 1970; Glynn and Hoffman 1971) and high concentrations of Na⁺_o (Garrahan and Glynn 1967a; Garay and Garrahan 1973), and includes a highly electrogenic Na⁺ deocclusion/release step (Bühler et al. 1991; Gadsby et al. 1993; Hilgemann 1994). The Na⁺_o dependence of this electroneutral exchange reaction has several similarities with the Na⁺_o dependence of the low affinity component of electrogenic exchange in Glu779Ala enzyme. However, two results suggest that electroneutral exchange and low Na⁺ affinity electrogenic exchange involve different reaction pathways. First, intracellular ADP inhibits low-affinity electrogenic exchange. Second, our previous data (Argüello et al. 1996) and the work of Vilsen 1995 show that high Na⁺ concentration promotes enzyme dephosphorylation, unlike electroneutral Na⁺–Na⁺ exchange. Thus, from our data, we conclude that the low Na⁺_o affinity component of electrogenic exchange occurs via enzyme dephosphorylation promoted by Na⁺_o binding at a Na⁺-specific site. Even so, we cannot determine whether the exchange reaction involves an E₁P(Na)₂- or E₂P(Na)₂-like conformation.

The second type of Na⁺–Na⁺ exchange reaction in wild-type enzyme uses Na⁺_o as a low-affinity K ⁺ substitute to stimulate enzyme dephosphorylation (Lee and Blostein 1980) and moves charge across the cell membrane (Forgac and Chin 1982; Cornelius and Skou 1985; Wuddel and Apell 1995). This electrogenic exchange occurs in the absence of ADP and is generally much slower than electroneutral Na⁺–Na⁺ exchange (Lee and Blostein 1980; Cornelius and Skou 1985). As a result, previous studies have not examined the V_M dependence of reactions involved in this transport mode in any detail. The present data show that the V_M dependence for Na⁺_o activation of high affinity Na⁺–Na⁺ exchange is similar to that for K ⁺_o activation of Na,K -pump current. The inability of intracellular ADP to influence this component of Na⁺–Na⁺ exchange is also expected if electrogenic exchange reactions follow a K ⁺-like dephosphorylation reaction pathway via an E₂P(Na)₂ conformation. Thus, Na⁺_o appears to be acting as an electrical as well as a biochemical congener of K ⁺_o in this sequence of exchange reactions.

The stoichiometry of Na⁺–Na⁺ exchange has previously been reported as 1:1 (Blostein 1983), 2:1 (Cornelius and Skou 1985), and varying with Na⁺ concentration (Forgac and Chin 1982). While we have no direct data on this point, our results are consistent with a 3:2 stoichiometry, at least for the low Na⁺_o affinity component of electrogenic exchange. First, if we assume that the enzyme extrudes three Na⁺ ions, an outward exchange current necessitates the number (n) of Na⁺ ions being transported inward to be less than three. Second, the Hill coefficient (_l) for the high-capacity, low-affinity component of electrogenic Na⁺–Na⁺ exchange is 2.0. Thus, n must be >1. Taken together, these data suggest that n must be an integer such that 1 < n < 3; i.e., n = 2.

To summarize, activation of the high affinity component of Na⁺–Na⁺ exchange shares some similarities with K ⁺_o activation of Na,K -pump current, analogous to the Albers-Post scheme (Glynn 1985). Activation of the low-affinity component has several similarities to Na⁺_o activation of electroneutral Na⁺–Na⁺ exchange, but is inhibited by intracellular ADP. These data would seem to indicate that Na⁺_o binding at a Na⁺-specific site promotes enzyme cycling. Overall, Na⁺_o-dependent activation of Glu779Ala enzyme turnover appears to occur at sites comparable with K ⁺_o and Na⁺_o sites in wild-type enzyme. The implication of this conclusion is that reaction kinetics in the mutant enzyme are altered, but, as pointed out above, without marked changes in the V_M dependence of extracellular ion-dependent reaction steps.

Other mutant enzymes showing a high rate of Na-ATPase activity have been identified recently (Argüello et al. 1999). These variant enzymes may also serve as beneficial model systems to study reactions that are otherwise difficult to examine in wild-type Na,K -ATPase.

Apparent V_M Independence of K ⁺_o-activated Currents in Na⁺_o-containing Solutions
Aside from the high level of Na⁺–Na⁺ exchange activity, the most surprising observation with Glu779Ala enzyme is the apparent V_M independence of K ⁺_o-activated currents measured in 148 mM Na⁺_o-containing solutions, even at K ⁺_o concentrations well below that which produces half-maximal current activation (Argüello et al. 1996). Glu779Gln enzyme also mediates Na,K -pump current that appears to be V_M-independent (over the entire range of V_M tested; –100 to +60 mV) at nonsaturating K ⁺_o concentrations; however, this enzyme does not have measurable Na⁺–Na⁺ exchange currents (Peluffo et al. 1997). Even so, the accompanying paper (Peluffo et al. 2000) shows that K ⁺ transport remains V_M dependent in Glu779Ala and Glu779Gln enzymes. Thus, one goal of this study was to find a reasonable explanation for these apparently conflicting data.

With wild-type Na,K -ATPase, a negative slope in I-V relationships of Na,K -pump current is observed at nonsaturating K ⁺_o concentrations. However, at high enough K ⁺_o, this negative slope is lost due to saturation of K ⁺ binding sites in the enzyme (Sagar and Rakowski 1994; Berlin and Peluffo 1997). A similar K ⁺-dependent saturation of ion binding sites can be ruled out as an explanation for the behavior of Glu779Ala enzyme because current amplitude varied widely at different K ⁺_o, as pointed out in the previous paragraph.

An alternative explanation for our experimental observations with Glu779Ala enzyme may lie in data suggesting that quaternary organic amines inhibit Na,K -pump current by interacting at two sites on the enzyme, one in and the other out of the membrane electric field (Berlin and Peluffo 1998). Previous reports have shown that quaternary amines inhibit Na,K -ATPase by acting as K ⁺_o antagonists (Kropp and Sachs 1977; Forbush 1988). Together, these data lead us to postulate that one K ⁺_o binding site in the Na,K -ATPase is located in the membrane dielectric and the other out of the dielectric.

To explain the lack of a negative slope in the I-V relationships for K ⁺_o-activated current in Na⁺_o-containing solutions, Na⁺_o must bind at the K ⁺_o site in the membrane dielectric with high enough affinity that this site is saturated with Na⁺_o and/or K ⁺_o at all K ⁺_o concentrations when [Na⁺] + [K ⁺] = 148 mM. Data showing that the high-affinity component of Na⁺–Na⁺ exchange has a K ⁰_0.5 for Na⁺_o of 13 mM are certainly compatible with this idea.

The implication of our postulate is that the substitutions Glu779Ala and Glu779Gln increase the relative affinity of the K ⁺ site in the membrane dielectric for Na⁺_o as compared with wild-type enzyme. This idea is consistent with Vilsen 1995, who concluded that rat Glu781Ala enzyme, equivalent to sheep Glu779Ala enzyme, had reduced selectivity between K ⁺ and Na⁺ for enzyme dephosphorylation.

Another feature of Glu779Ala and Glu779Gln enzymes is the lack of positive slope in the I-V relationships for Na,K -pump current in Na⁺_o-containing solutions (Argüello et al. 1996; Peluffo et al. 1997). Wild-type Na,K -ATPase displays similar behavior in Na⁺_o-free solutions (Nakao and Gadsby 1989; Sagar and Rakowski 1994; Berlin and Peluffo 1997; Peluffo et al. 2000). Thus, an explanation for this lack of positive slope in the I-V relationship of variant enzymes could be that the apparent affinity for Na⁺_o binding to E₂P is greatly decreased with Glu779Ala and Glu779Gln substitutions. In fact, the Na⁺ binding affinity need not actually change. Instead, a decrease in apparent affinity for the inhibitory action of Na⁺_o could just reflect the fact that Na⁺_o can also promote forward enzyme cycling in variant enzymes.

Effect of Glu779Ala on the V_M Dependence of Extracellular Ion Binding Reactions
An alternative explanation for the apparent V_M independence of Na,K -pump current observed in Na⁺-containing solutions is that the V_M dependence of extracellular ion binding reactions are altered by mutations at Glu779. Our experimental data alone do not allow us to judge whether this explanation is reasonable. Thus, to address this point, computer simulations were developed to qualitatively reproduce our experimental observations with Glu779Ala enzyme using a reaction scheme that was as simple as possible. Previously published pseudo three-state (Sagar and Rakowski 1994) and pseudo four-state models (Wang et al. 1996) could not account for the biphasic response of Na⁺–Na⁺ exchange current to changes in Na⁺_o concentration, nor the V_M independence of K ⁺_o-activated currents discussed above (see Argüello et al. 1996; Peluffo et al. 1997). Both of these observations could be reproduced by a branched pseudo five-state reaction scheme that included (a) a reaction pathway in which enzyme turnover was promoted by Na⁺_o binding at a Na⁺-specific site, and (b) a reaction pathway with two nonidentical K ⁺_o binding sites, one located in the membrane dielectric and the other out of the membrane electric field. To reproduce the steepness of the negative slope in the I-V relationships under Na⁺_o-free conditions (see Figure 3 B in Peluffo et al. 2000), we had to assume an ordered binding scheme (Forbush 1987) with the K ⁺_o binding first at the site located outside the membrane dielectric. Furthermore, Na⁺ binding to K ⁺ sites also promoted enzyme turnover.

The pseudo five-state model could not reproduce the lack of Na⁺_o-dependent shift in the I-V relationships for Na⁺–Na⁺ exchange current at higher Na⁺_o concentrations (Fig. 4). To account for this observation, V_M dependence in the Na⁺-specific reaction pathway was assigned to a step following Na⁺ loading of the enzyme. This step could represent ion occlusion, analogous to models of the Na/Ca exchanger adopted by Matsuoka and Hilgemann 1992, or enzyme interconversion between E₁ and E₂ conformations. Although our results cannot distinguish between these alternatives, enzyme interconversion in wild-type enzyme has been shown to display little V_M dependence (Wuddel and Apell 1995). Thus, we prefer the former explanation, which would suggest that Glu779Ala mutation changes reaction kinetics associated with Na⁺_o occlusion, rather than the latter, which would imply a major change in the identity of charge-moving reaction steps. Assignment of V_M dependence to such a reaction step necessitated that we propose a branched pseudo six-state model to simulate ion transport by Glu779Ala enzyme.

The reaction steps comprising this pseudo six-state model are shown in Fig. 8 A. For the K ⁺-sensitive clockwise reaction pathway (i.e., the Albers-Post scheme; Glynn 1985), we assume that transport stoichiometry is the same as wild-type enzyme, whether enzyme turnover is stimulated by K ⁺_o or Na⁺_o. As discussed above, our data for the low Na⁺_o affinity component of Na⁺–Na⁺ exchange are also consistent with a 3:2 stoichiometry. Each reaction branch is also modeled with an irreversible step that lumps together all reactions subsequent to extracellular ion binding/occlusion (Hansen et al. 1981). This assumption is consistent with our experimental conditions (i.e., high intracellular Na⁺ and ATP concentrations), which tend to strongly favor forward enzyme cycling. In addition, the assumption simplifies the analytical solution of the model (see ).

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Simulations of ion transport by Glu779Ala enzyme using a pseudo six-state model for Na,K -ATPase. (A) Reaction scheme. Na+o-specific binding steps occur between states E2 and E3, while K +o binding reactions, which can also involve Na+o, as indicated by "(Nao)," occur between states E2, E5, and E6. All reactions are reversible except those indicated by the single arrows (). Greek characters adjacent to the arrows represent forward (i) and backward (βi) rate constants. (B) Wild-type–like behavior. K +o-activated Na,K -pump current was simulated in the presence of Na+o. K +o binding to the site located in the membrane dielectric is assumed to occur to the exclusion of Na+o binding. Potassium concentrations (bottom to top): 0.2, 0.5, 1, 2, 5, 10, and 20 mM. [Na+]o + [K +]o = 148 mM. (C) Glu779Ala-substituted enzyme. K +o-activated Na,K -pump current was simulated in the presence of Na+o. Na+o and K +o are assumed to bind to the site in the membrane dielectric with comparable affinities. Potassium concentrations (bottom to top): 1, 2, 5, 10, 20, and 50 mM. [Na+]o + [K +]o = 148 mM. (D) Glu779Ala-substituted enzyme. K +o-activated Na,K -pump current was simulated in the absence of Na+o. Potassium concentrations (bottom to top): 0.2, 0.5, 1, 2, 5, 10, and 20 mM. (E) Glu779Ala-sub–stituted enzyme. Na+o-activated Na+–Na+ exchange current was simulated in K +-free solution. Sodium concentrations (bottom to top): 1, 3, 6, 12, 25, 50, 75, and 148 mM. (Inset) Data from Fig. 5 at –100 (•), 0 (), and +60 () mV are replotted along with the results of the simulations (solid curves). The following parameters had the same value for all simulations: Hill coefficients, 2 = 2.2, β1 = 1.0; fractional electrical distances, β1 = 0.7, 6 = 0.35, 3/β3 = 0.8; n = 1.21 · 10–7 mol/cm2; T = 310°K .

Taken as a whole, our simulations suggest that the substitution Glu779Gln, which removes the carboxyl moiety from the side chain, has two major effects. The first effect is to change the relative Na⁺_o and K ⁺_o affinities of a K ⁺_o binding site located in the membrane dielectric so that the site is saturated in 148 mM Na⁺_o-containing solutions. The second effect is a decrease in the apparent affinity for Na⁺_o to inhibit forward enzyme cycling. In addition, the substitution Glu779Ala, which also removes side chain bulk, promotes enzyme turnover after Na⁺_o binds to the Na⁺-specific site and/or K ⁺-binding sites. In wild-type enzyme, this reaction must be strongly inhibited to ensure efficient exchange of Na⁺ and K ⁺ across the cell membrane. The structure of the side chain at residue 779 must, therefore, contribute to the high threshold energy that normally prevents electrogenic Na⁺–Na⁺ exchange from occurring at a high rate.

Equations describing the reaction scheme in 8 A were derived for steady state conditions using the King and Altman method (Segel 1975). This derivation yielded the following expression for Na,K -ATPase-mediated current as a function of membrane potential and extracellular ion concentration:

where [X] represents Na⁺_o and/or K ⁺_o concentration, F is the Faraday constant, n is the total amount of enzyme per square centimeter of membrane area, and

in which pseudo first-order and/or V_M-dependent rate constants were defined as follows:

where _β1 and ₂ are the Hill coefficients for those Na⁺_o-binding reactions of uncertain stoichiometry; _β1, _3/β3, and ₆ are the fractional electrical distances for the respective V_M-dependent reactions; and U, the reduced membrane potential, equals F V_M/RT. In the case of the rate constants describing the ion-independent V_M-dependent transition, ₃ and β₃, the membrane potential dependence was introduced via a symmetric Eyring barrier (Läuger and Stark 1970).

Steady state ion transport rate (i.e., current) was simulated using Mathcad 8 Professional software (Mathsoft) run on a personal computer. In these simulations, explicit interactions between Na⁺ and K ⁺ at extracellular ion binding sites of Glu779Ala enzyme were not modeled. Instead, rate constants were varied to account for possible interactions at ion binding sites. The K ⁺_o binding site for Glu779Ala enzyme located in the membrane dielectric was assumed to bind Na⁺_o and K ⁺_o with the same affinity, consistent with the suggestion of Vilsen 1995, so that the enzyme could effectively "see" the sum of the concentration of both ions.

In the simulations, transformation of I-V relationships from a wild-type–like (8 B) to a Glu779Ala-like (8 C) behavior in the presence of extracellular Na⁺ required a 100-fold decrease in the rate constant for inhibitory Na⁺_o rebinding, β₁ (see 8 A), together with a twofold increase in the dissociation constant for binding of K ⁺_o to the V_M-independent site in the pump (β₅/₅), as shown in . Furthermore, the K ⁺_o binding site located in the membrane dielectric was assumed to have zero Na⁺_o affinity in wild-type enzyme; however, this site was assumed to bind Na⁺_o and K ⁺_o with equal affinity in Glu779Ala enzyme, consistent with the finding that Na⁺_o activates the "K ⁺-like" high-affinity, low-capacity component of electrogenic Na⁺–Na⁺ exchange with a K ⁰_0.5 of 10 mM. With these constraints, the lack of V_M dependence in the I-V relationships for Glu779Ala enzyme reported in solutions containing Na⁺_o and nonsaturating K ⁺_o concentrations (Argüello et al. 1996) could be well reproduced (8 C).

To reproduce the V_M and K ⁺_o dependence of Na⁺–K ⁺ exchange by Glu779Ala under Na⁺_o-free conditions (8 D), the forward rate constant for K ⁺_o binding to the V_M-independent site (₅) was increased 200-fold, as compared with the wild-type enzyme, with only minor changes in the other three rate constants involved in K ⁺_o binding/release (β₅, ₆, and β₆, see ).

Simulation of Na⁺–Na⁺ exchange electrical behavior by Glu779Ala variant enzyme (8 E) required a large increase in the forward rate constant for Na⁺_o binding (₂) with respect to the wild-type enzyme, so that a significant fraction of total enzyme would cycle through the counterclockwise branch of the reaction scheme shown in 8 A. The apparent dissociation constant for Na⁺_o binding (β₂/₂) was set to 0.4 M. To simulate the high-affinity, low-capacity Na⁺–Na⁺ exchange component (clockwise reaction pathway in 8 A), Na⁺_o was assumed to act as a low-affinity K ⁺_o congener. Thus, values of the forward (₅, ₆) and backward (β₅, β₆) rate constants for Na⁺_o binding were decreased from those used to simulate K ⁺_o binding to wild-type Na,K -pump. Finally, the low-capacity characteristic of this Na⁺–Na⁺ exchange current component was obtained by reducing fivefold the value of the rate constant for the irreversible step (₇).

	ACKNOWLEDGMENTS

 
The authors thank the excellent technical assistance of Ms. Palak Raval-Nelson and Marguarita Schmid.

This work was supported by a Grant-in-Aid from the American Heart Association (J.R. Berlin), a postdoctoral fellowship from the Southeastern Pennsylvania affiliate of the American Heart Association (R.D. Peluffo), and grants HL03373 (J.M. Argüello), GM57253 and HL43712 (J.R. Berlin), and HL28573 (J.B Lingrel) from the National Institutes of Health.

Submitted: 10 November 1999
Revised: 29 March 2000
Accepted: 12 May 2000

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