Correspondence to: Joshua R. Berlin, Department of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, 185 S. Orange Avenue, Newark, NJ 07103. Fax:973-972-7950 E-mail:berlinjr{at}umdnj.edu.
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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 (3148 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. 116:4759). 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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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 (VM) to affect current amplitude is observed at widely varying K +o concentrations, including those well below the concentration for half maximal current activation (
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 VM independent over a broad range of K +o and VM in extracellular Na+ (Na+o)-containing solutions. However, unlike Glu779Ala enzyme, electrogenic Na+Na+ exchange was not measurable (
Membrane potential-dependent behavior of the Na,K -ATPase is due to the electrogenicity and kinetics of reactions controlling the ion transport cycle (
This study demonstrates that Na+o-dependent reactions of Glu779Ala enzyme are highly VM 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 (
Using data in this and previous studies (
Finally, the VM-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.
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METHODS |
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Mutagenesis, transfection, and cell culture protocols were similar to the previous paper (
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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 3060% of maximal Na,K -ATPase activity, a level that is much higher than wild-type enzyme (
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Electrogenic Na+Na+ Exchange Current
Our previous work suggested that Na-ATPase activity can be observed as electrogenic Na+Na+ exchange in voltage-clamped HeLa cells expressing Glu779Ala enzyme (
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.
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The voltage dependence of Na+Na+ exchange current was examined by stepping the membrane potential from -100 to +60 mV in 10-mV increments during superfusion with 1 µM and 10 mM ouabain-containing solutions. Typical currentvoltage relationships for two selected Na+o concentrations, 12.5 and 148 mM, show that increasing ouabain concentration resulted in an inward shift of current at all potentials (Fig 3), as would be expected for an exchange process that moved net positive charge out of the cell. As in Fig 2, the amplitude of the ouabain-sensitive current was larger at the higher Na+o concentration. In addition, current amplitude decreased at positive VM with both Na+o concentrations. Na+Na+ exchange current was calculated as a difference current at each VM by subtracting current recorded in the presence of 10 mM ouabain from that recorded during superfusion with 1 µM ouabain.
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Fig 3 also shows that, as Na+o concentration was decreased, the slope conductance of the cell decreased. This change probably reflects the decreased concentration of permeant ions in the extracellular solution. The increased membrane resistance was accompanied by decreased current noise so that current densities less than 0.1 pA/pF (i.e., 35 pA in a typical cell) could readily be measured.
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 VM with current density decreasing as VM became more positive. The voltage dependence of ouabain-sensitive difference current in the presence of Na+o contrasts with the relative VM independence of K +o-activated current observed in 148 mM Na+o-containing solution (
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The negative slope in the I-V relationship could be analogous to the negative slope of K +o-activated Na,K -pump current observed in Na+o-free solutions with wild-type (
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 VM, we did not investigate this point further.
To examine the VM dependence of current more carefully, current density at selected VM 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 VM, current activation was a biphasic function of Na+o. Even at positive VM, 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 VM was fitted with a two-component Hill equation,
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(1) |
which included exchange current activated with relatively high (h) and low (l) affinities for Na+o. Maximum overall current (i.e., Imax,h + Imax,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. Equation 1 was fitted to the data at all VM. 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 VM became more positive, a result that suggests both current components are VM dependent.
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The Hill coefficient for the low affinity current component (l) was not dependent on VM. As a result,
l at all VM were averaged to yield a value of 2.2 ± 0.6 (n = 17), an indication that the activation of current at higher Na+o concentrations displayed positive cooperativity; i.e., more than one Na+ is involved. This fitting procedure also showed that Imax,h was considerably smaller than Imax,l, consistent with the high affinity current component having a lower capacity for ion transport.
Given the apparent ability of Na+o to act as a K +o-like congener, we first attempted to analyze the VM-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 Equation 2 in x) dissipated during Na+o-dependent activation can be estimated by the following:
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(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 VM. As pointed out in
Fitting Equation 2 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 Equation 2 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 Equation 2 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 Equation 1 in the preceding paper (
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(3) |
where K l0 is K l at 0 mV, B is the product of VM-independent rate constants, and l
l and
i
i 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, Equation 3 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 (
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 (
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 (
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DISCUSSION |
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In this and the accompanying paper (
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 (
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;
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 (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 (
The low-affinity component of Na+Na+ exchange current is highly VM-dependent and accounts for 76% of calculated maximal Na+Na+ exchange current at -100 mV. Using Equation 3, which is based on a pseudo three-state scheme for the Na,K -ATPase (80% of the membrane electric field. It is interesting that the VM dependence of this reaction appears to be quite similar to that for Na+o rebinding in wild-type enzyme (
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 00.5 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 (
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 (
The stoichiometry of Na+Na+ exchange has previously been reported as 1:1 (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 (
Other mutant enzymes showing a high rate of Na-ATPase activity have been identified recently (
Apparent VM 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 VM 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 (
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 (
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 (
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 00.5 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
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 (
Effect of Glu779Ala on the VM Dependence of Extracellular Ion Binding Reactions
An alternative explanation for the apparent VM independence of Na,K -pump current observed in Na+-containing solutions is that the VM 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 (
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, VM 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
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;
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Using this model, simulations were performed for: (a) wild-type and (b) Glu779Ala Na,K -pump current in the presence of Na+o, (c) Glu779Ala Na,K -pump current in the absence of Na+o, and (d) Glu779Ala Na+Na+ exchange current in K +-free solution (see Appendix). The simulated I-V relationships, obtained using the rate constants listed in Table 1 (see Appendix) are displayed in Fig 8BE. In all cases, simulated maximum current levels (Imax) were consistent with their experimental counterparts. The values and VM dependence of K 0.5 calculated from the simulated I-V relationships were similar to those found in the experiments. Even the biphasic dependence of Na+Na+ exchange current on Na+o was reproduced (Fig 8 E, inset).
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An important conclusion derived from these simulations is that wild-typelike and Glu779Ala electrical behaviors under all ionic conditions could be modeled by modifying rate constants for ion binding reactions while keeping the fractional electrical distances for VM-dependent reactions unchanged. Thus, our simulations suggest that the unique VM dependence of ion transport by Glu779Ala enzyme can be explained by changes in reaction kinetics without the need to invoke alterations in the VM dependence of extracellular ion binding reactions.
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.
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Footnotes |
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Portions of this work were previously published in abstract form (Peluffo, R.D., J.M. Argüello, J.B Lingrel, and J.R. Berlin. 1998. Biophys. J. 74:A191).
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Acknowledgements |
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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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Appendix |
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Equations describing the reaction scheme in Fig 8 A were derived for steady state conditions using the King and Altman method (
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 VM-dependent rate constants were defined as follows:
where ß1 and
2 are the Hill coefficients for those Na+o-binding reactions of uncertain stoichiometry;
ß1,
3/ß3, and
6 are the fractional electrical distances for the respective VM-dependent reactions; and U, the reduced membrane potential, equals F VM/RT. In the case of the rate constants describing the ion-independent VM-dependent transition,
3 and ß3, the membrane potential dependence was introduced via a symmetric Eyring barrier (
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
In the simulations, transformation of I-V relationships from a wild-typelike (Fig 8 B) to a Glu779Ala-like (Fig 8 C) behavior in the presence of extracellular Na+ required a 100-fold decrease in the rate constant for inhibitory Na+o rebinding, ß1 (see Fig 8 A), together with a twofold increase in the dissociation constant for binding of K +o to the VM-independent site in the pump (ß5/5), as shown in Table 1. 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 00.5 of
10 mM. With these constraints, the lack of VM dependence in the I-V relationships for Glu779Ala enzyme reported in solutions containing Na+o and nonsaturating K +o concentrations (
To reproduce the VM and K +o dependence of Na+K + exchange by Glu779Ala under Na+o-free conditions (Fig 8 D), the forward rate constant for K +o binding to the VM-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 (ß5,
6, and ß6, see Table 1).
Simulation of Na+Na+ exchange electrical behavior by Glu779Ala variant enzyme (Fig 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 Fig 8 A. The apparent dissociation constant for Na+o binding (ß2/
2) was set to 0.4 M. To simulate the high-affinity, low-capacity Na+Na+ exchange component (clockwise reaction pathway in Fig 8 A), Na+o was assumed to act as a low-affinity K +o congener. Thus, values of the forward (
5,
6) and backward (ß5, ß6) 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).
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