Correspondence to: Paul De Weer, Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085. Fax (215) 573-5851; E-mail:deweer{at}mail.med.upenn.edu.
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Abstract |
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The steady-state voltage and [Na+]o dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K+-free seawater; ATP- and Na+-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated 42K+ efflux to reverse pump current, Ipump (both defined by sensitivity to dihydrodigitoxigenin, H2DTG), scaled by Faraday's constant, was -1.5 ± 0.4 (n = 5; expected ratio for 2 K+/3 Na+ stoichiometry is -2.0). Steady-state reverse pump current-voltage (Ipump-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various Vm to H2DTG or from the difference between membrane I-V relationships obtained by imposing Vm staircases in the presence or absence of H2DTG. With the second method, we also investigated the influence of [Na+]o (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse Ipump-V relationship. The reverse Ipump-V relationship is sigmoid, Ipump saturating at large negative Vm, and each doubling of [Na+]o causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state 22Na+ efflux during electroneutral Na+/Na+ exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100103). This model predicts valence z = n, where n (1.33 ± 0.05) is the Hill coefficient of Na binding, and
(0.61 ± 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na+/Na+ exchange modes only with additional assumptions that render them less likely.
Key Words: Na,K-ATPase, electrogenicity, active transport, kinetics, modeling
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INTRODUCTION |
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The electrogenic sodium pump or Na,K-ATPase exports three Na ions and imports two K ions for each molecule of ATP hydrolyzed. According to the generally accepted Albers-Post model (
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Information on charge-translocating steps in a transporter's reaction cycle can be obtained from the voltage dependence of the turnover rate of its various modes of operation (for reviews see
Much evidence shows that extracellular Na+ and K+ both interact with the Na/K pump at sites within the membrane's electric field (Na/Na) declines at positive potentials, but, upon membrane hyperpolarization rises along an apparently saturating sigmoid curve. Furthermore, activation of Na+/Na+ exchange by Na+o and by hyperpolarization are kinetically equivalent (
Na/Na-V) curve is simply shifted laterally when [Na+]o is altered. Both characteristics point to a voltage sensitivity related to the external Na+ rebinding step, which therefore must be influenced by the membrane field (
A third mode of operation of the sodium pump, besides electrogenic forward Na+/K+ transport and electroneutral Na+/Na+ exchange, is electrogenic K+/Na+ transport in which the cycle runs backwards to generate both ATP (
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MATERIALS AND METHODS |
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Voltage Clamp and Internal Dialysis
Membrane current and unidirectional 42K+ tracer efflux were measured in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, using methods previously described (75 µm in diameter) were inserted through the other end. A conventional voltage clamp circuit measured membrane current, and two ancillary voltage clamp circuits prevented longitudinal stray current flow between the center pool and adjacent guard pools. The cytoplasmic composition was maintained and, in some cases, 42K+ was introduced inside the axon by perfusing the porous intracellular capillary (1.4 µl/min) with appropriate solutions. To measure 42K+ efflux, the artificial seawater superfusing (2 ml/min) the axon in the center pool was collected in scintillation counting vials. Initial experiments were carried out near 17°C; but, to increase sodium pump turnover rate and hence signal magnitude, the temperature was raised to
22°C in later experiments (see Fig 4 and Fig 7). A 3060 min period of dialysis (to allow for equilibration) preceded data collection in all experiments.
Solutions
External solutions were K+-free artificial seawater typically containing (in mM): 400 Na isethionate, 75 Ca sulfamate, 5 Tris HEPES, pH 7.7, 1 3,4-diaminopyridine (DAP), 0.1 Tris EDTA, and 0.2 µM tetrodotoxin (TTX). In 200 mM-Na+ seawater N-methyl-D-glucamine (NMG) sulfamate replaced half of the Na isethionate. In some early experiments, chloride replaced the isethionate and sulfamate. The internal dialysis solutions were Na+-free and typically contained (in mM): 160 K+, 100 glycine, 50 phenylpropyltriethylammonium (PPTEA) sulfate, 2.5 BAPTA, 40 Mg HEPES, 5 dithiothreitol, 25 ADP, and 25 orthophosphate, pH
7.5; the principal anion was HEPES. In some early experiments, EGTA replaced BAPTA, MgSO4 or MgCl2 replaced Mg HEPES, and 2025 mM L-arginine (a substrate for arginine kinase) sulfate or chloride, 12.5 mM Li5-diadenosine pentaphosphate (an inhibitor of adenylate kinase), and 50100 µM Na atractyloside (an inhibitor of mitochondrial ATP/ADP transfer) were added to minimize ATP accumulation within the axon. The osmolality of these internal solutions was 930940 mOsm/kg, and that of the external solutions was 920930 mOsm/kg. To extend the experimental [Na+]o range, hypertonic (1,7801,790 mOsm/kg) internal and external solutions were used in some cases. In hypertonic dialysis fluid, 130 mM NMG HEPES replaced glycine. One hypertonic artificial seawater matched the typical composition above except that it contained 800 mM Na isethionate, and the other contained 800 mM NMG sulfamate instead. Intermediate (200 and 400 mM) Na concentrations were obtained by mixing.
External TTX and DAP, and internal PPTEA, minimized nonpump Na and K channel currents (90% by 10 µM H2DTG in 400 mMNa+, K+-free seawater (
94% by 100 µM H2DTG in Na+-free seawater (
0.1% vol/vol) did not affect the membrane current under our conditions (see Fig 5 A). Ouabain was used at 100 µM, from a 10-mM stock solution in the appropriate artificial seawater. DAP was added directly to artificial seawater, and TTX from a 1-mM aqueous stock.
Correction for Baseline Drift
In two instances the shapes of several I-V plots obtained under different conditions and/or from different axons needed to be rigorously compared (two experiments on a single axon are compared in Fig 6 C, and numerous experiments on two dozen axons are compared in Fig 7A and Fig B). To that end, it was necessary to correct each individual "raw" H2DTG (or ouabain) difference I-V curve for any inadvertently included spontaneous baseline conductance drift. Baseline drift was estimated from "time only" differences (typically two before, and two after, exposure to the pump blocker) between consecutive I-V plots recorded at time intervals matching that between the I-V plots obtained just before and shortly after addition of the pump blocker (illustrated in Fig 4). To avoid the sampling noise that would result from point-by-point correction, the "time only" difference I-V plots were individually fit with second-degree polynomials, a time-weighted average of which was then subtracted from the raw signal difference to yield the drift-corrected H2DTG-sensitive I-V plot. To obviate time weighting, the six experimental I-V records were generally (as in Fig 4) evenly spaced. The drift correction was usually small (6% of Ipump at -60 mV in most axons.)
Global Least-Squares Fit of Access Channel Model to the Data of Fig 7
Our access-channel model (see DISCUSSION) yields an S-shaped expression for the voltage and [Na+]o dependent reverse pump current of the form:
(where Vm is membrane potential and F, R, and T have their usual meaning), such that reverse Ipump vanishes at extreme positive potentials and reaches Imaxpumpat extreme negative potentials. This expression can be interpreted either as a Boltzmann partition equation with [Na+]o-dependent midpoint or as a Hill approximation with fixed Hill coefficient n but voltage-dependent affinity for external Na+. K00.5 is the apparent dissociation constant of a Na+-binding site at the bottom of an access channel in the absence of transmembrane field, and is the access channel's depth as a fraction of electrical distance across the membrane, measured from the external solution.
To combine and compare multiple data sets from different axons (see Fig 7), the following procedure was adopted. All I-V plots, individually corrected for baseline drift as described, were simultaneously fit, in a global least-squares procedure () common to all experiments; (2) a Hill coefficient n common to all experiments; and (3) two values for the apparent Na affinity in the absence of membrane potential (K00.5): one value shared by all normotonic experiments (see Fig 7 A) and another shared by all hypertonic experiments (see Fig 7 B). All theoretical curves in Fig 7 are drawn for 22°C (where RT/F = 25.34 mV) with these common parameters, and with normalized Imaxpump= -1 and no offset at positive voltages. The drift-corrected data were offset to zero at large positive voltages, normalized to -1 at large negative voltages, grouped by experimental protocol, and averaged. Symbols represent averages ± SEM. Lognormal statistics were used to calculate the standard error of ratios.
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RESULTS |
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Reverse Pump Stoichiometry: Simultaneous Measurements of H2DTG-sensitive Current and 42K+ Efflux
Fig 1 shows representative results from one of five axons engaged in reverse pumping in which H2DTG-sensitive current and 42K+ efflux were measured simultaneously at -60 mV. Since H2DTG is a specific Na/K pump inhibitor without additional effects (but see below) the small H2DTG-induced outward shift of the holding current (I = 0.055 µA cm-2 in Fig 1 A) must reflect abolition of the inward current generated by the backward-running Na/K pump. H2DTG washout allows repetition of this type of experiment. The mean
I of three successive trials on the axon of Fig 1 A was 0.056 ± 0.002 µA cm-2. Fig 1 B shows the average of the corresponding three 42K+ efflux determinations; mean H2DTG-sensitive K efflux (-
) was 1.04 ± 0.07 pmol cm-2 s-1 in this axon, yielding a value of 1.79 ± 0.22 for the ratio -F
/
(where F is Faraday's constant), which is compatible with the value of 2.0 expected for 2 K+/3 Na+ transport stoichiometry. The mean values of
I and
for all five axons were 0.106 ± 0.011 µA cm-2 (n = 18) and -1.46 ± 0.19 pmol cm-2 s-1 (n = 16). The (geometric) mean of the individual -F
/
I ratios was 1.5 ± 0.4, not significantly (P > 0.2) different from 2.0. The correspondence of H2DTG-sensitive current and flux argues that both reflect backward cycling of the Na/K pump.
Membrane Potential Effect on Reverse Pump Current
We used two procedures to estimate the voltage dependence of reverse pump current magnitude. The first determined the magnitude of H2DTG-sensitive current at various holding potentials by repeated exposures of an axon to H2DTG with intervening periods of washout. Fig 2 shows successive H2DTG-induced holding current shifts in a single axon. The records in the first column (at the reference holding potential of -40 mV) show a steady decline of reverse pump current magnitude with time. The I measurements at other holding potentials, therefore, were corrected for this rundown by exponential interpolation (extrapolation for final measurement). The H2DTG-induced
I is large at negative potentials, negligible at about +10 mV, and reversed at +30 mV. Data from 14 axons (including that of Fig 2) are summarized in Fig 3 A, which plots the magnitude of the abolished backward-running pump current. In six of these axons, bracketed data allowed correction for rundown and normalization to the interpolated magnitude of the H2DTG-induced
I at -40 mV (Fig 3 B). The results show that reverse pump current magnitude increases monotonically with hyperpolarization throughout the voltage range examined. The small outward current at +30 mV is probably caused by small variations of [K+] in the extracellular space, as will be discussed later.
Reverse Pump Current-Voltage Relationship
In the second method, illustrated in Fig 4, we recorded entire steady-state I-V curves in the absence and presence of H2DTG (or ouabain), and obtained the reverse pump I-V relation by difference, subject to correction for any baseline drift. This method, though unsuited for simultaneous measurement of 42K+ efflux, more accurately renders the shape of the I-V relationship and (being rapid) is much less affected by rundown. Fig 4 A shows a sequence (af) of six identical 48-step down-up-down membrane potential staircases (1 s per 5-mV step) imposed from a holding potential of -30 mV, before and during exposure to 100 µM H2DTG. Fig 4 B shows the resulting changes in membrane current, as well as the outward shift upon application of H2DTG (between c and d) reflecting abolition of inwardly directed reverse pump current. The repeated I-V measurements allowed accurate assessment of any spontaneous drift in the shape of the membrane I-V curve before and/or after H2DTG addition.
Fig 4 C shows four (of the six) corresponding steady-state membrane I-V relationships, two (b and c; circles) before and two (d and e; squares) during exposure to H2DTG, obtained by plotting the current recorded near the end of each 1-s staircase step against step voltage. The measured currents reflect a true steady state as there was negligible hysteresis in the I-V plots, the same steady-state current being obtained at a given voltage during ascending and descending limbs of the staircase. The overlapping of open and filled symbols in Fig 4 C indicates only modest drift with time. The difference (Fig 4 D, c and d; closed circles) between records just before (c) and shortly after (d) H2DTG addition gives a raw H2DTG-sensitive I-V plot, so qualified because it is not corrected for any unrelated, spontaneous baseline drift that occurred during the 5-min interval between c and d. Even without baseline drift correction, the voltage dependence of H2DTG-sensitive current is clearly sigmoid, with inward current apparently saturating at large negative potentials.
Four estimates of how much the baseline drifted during an equivalent time interval are given by difference I-V relationships obtained in the absence (a minus b and b minus c) and presence (d minus e and e minus f) of H2DTG; one of each is shown in Fig 4 D. The raw H2DTG-sensitive I-V plot, if necessary (see MATERIALS AND METHODS), can be corrected for this here negligibly small baseline drift. For any Vm, the drift-corrected H2DTG-sensitive current is given by the distance between the raw H2DTG-sensitive plot and the background drift plot.
Evidence that H2DTG-induced Shifts in Holding Current Represent Na/K Pump Current
We have previously established (
As in the case of forward Na/K pump current (
H2DTG- and Ouabain-sensitive Currents
Current-voltage relationships of the backward-running pump obtained by H2DTG or ouabain inhibition are compared in Fig 6. Fig 6 A shows (closed circles) the mean raw H2DTG difference I-V relationship obtained from seven measurements on four axons. (The absolute current magnitudes were sufficiently similar to warrant simple averaging without normalization.) Open circles show the average ± SEM of the corresponding seven baseline drift estimates. Fig 6 B shows the analogous data for ouabain, also simply averaged, obtained from four axons. There are no obvious differences between the two sets except perhaps for the small outward current at positive potentials in the case of H2DTG.
Since ouabain binds irreversibly to the Na pump of squid giant axons, measurements of ouabain-sensitive difference currents were usually performed after some other protocol(s) had been completed. At the concentration of the reversible blocker H2DTG used (100 µM), and under our experimental conditions, its difference signal should be 94% of that of ouabain (
Influence of Extracellular [Na+] on the Backward-running Sodium Pump I-V Curve
The saturating sigmoid shape of the reverse pump I-V relationship, tending to a constant maximal reverse pump current Imaxpumpat extreme negative potentials, is predicted by our model (see DISCUSSION) which features an external high field access/release channel. A key property of the access channel models is that alterations in membrane potential and alterations in concentration of a charged reagent (here, external Na+) are kinetically equivalent (
These predictions were verified by our findings. First (data not shown), when repeated inward pump I-V curves were obtained on a single axon and fit with Boltzmann partition functions, the (extrapolated) maximal pump current magnitudes Imaxpumpwere indistinguishable (except for exponential rundown) whether the axon, if hypertonic, was bathed in 800, 400, or 200 mM Nao or, if normotonic, in 400 or 200 mM Nao. Second, Fig 7 shows that, for both normal tonicity solutions (in which [Na+]o of 400 and 200 mM can be compared; Fig 7 A) and hypertonic solutions (that permit raising [Na+]o to 800 mM; Fig 7 B), the parallel shifts are identical (29 mV) for each twofold change of external Na+ concentration. The magnitude of this shift reflects the fraction of the membrane's electric field dropped along the postulated access channel, = 0.61 ± 0.03 (see DISCUSSION). The uniform apparent valence (z = n
= 0.80) of all five Boltzmann partition curves reflects, besides the common
already mentioned, a common molecularity (Hill coefficient) value of n = 1.33 ± 0.05. The midpoint voltages of the curves reflect values for the apparent Nao affinity in the absence of transmembrane potential (K00.5) of 715 ± 37 mM for normotonic, and 1,065 ± 80 mM for hypertonic axons.
In practice, the simultaneous global fit to all the data (normotonic and hypertonic) shown in Fig 7 was preceded by one (not shown) in which normotonic and hypertonic data were fit separately. After verifying that the refined least-squares parameters did not differ significantly between the two groups, we proceeded with the global fit as described in MATERIALS AND METHODS.
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DISCUSSION |
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Reversal of the Na/K Pump
Reliability of Reverse Pump Current Measurements
The technical difficulties of accurately measuring the small inward current and K+ efflux generated by the backward-running Na/K pump have been largely overcome. During reverse pumping, K ions are extruded into an extracellular restricted-diffusion space (
Kinetic Model of the Na/K Pump
The kinetics of the Na/K pump are well described by the Albers-Post model (
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It is firmly established that positive charge is carried outward across the membrane during the Na+-translocating part of the forward Na/K pump cycle, predominantly in steps that occur late in the Na+ hemicycle (for reviews see
Two Modes: Na+/Na+ Exchange and Reverse Pumping
Because the ADP-requiring Na+/Na+ exchange mode and the reverse pumping mode likely have reaction steps in common, and because we have identified (
Voltage Dependence of Na+/Na+ exchange
In the discussion that follows, we will analyze the steady-state voltage dependence of the reverse pumping mode in light of our previous findings on the Na+/Na+ exchanging mode. We have shown ( 50 mM, the voltage sensitivity of Nai+ binding mentioned earlier is negligible for practical purposes). A formal interpretation of the observed behavior is that step 2 takes place over an Eyring barrier that is positioned highly asymmetrically within the membrane electrical field (our data required
95% asymmetry; see
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(1) |
where k'-2 is the corresponding voltage-insensitive intrinsic higher-order rate coefficient, is the fraction of the membrane field dropped along the access channel measured from the external solution edge, and n is the empirical Hill coefficient (apparent molecularity) of external Na+ binding. In this simple physical model, the lateral voltage shifts caused by doubling of [Na+]o are defined by
V = (RT/
F)ln2, and the apparent valence of the Boltzmann partition function is defined as z = n
.
If k-2 is effectively (over the accessible voltage range) the sole voltage sensitive rate coefficient in the Na+/Na+ exchange reaction scheme of Fig 9 A, then this pseudo-4-state scheme may be further reduced to the pseudo-2-state Na+/Na+ exchange scheme of Fig 9 C, without loss of kinetic information with regard to steady-state dependence of exchange rate on Vm and [Na+]o. (For a general proof that any multistep unbranched reaction cycle with a single voltage-sensitive step can be reduced to a pseudo-two-state model for the purpose of describing the voltage sensitivity of steady-state currents or turnover rates see Na/Na = f(Na+]o) K0.5 or
Na/Na = f(Vm):
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(2a) |
(a Hill equation with voltage-sensitive apparent K0.5), or
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(2b) |
(a Boltzmann equation with [Na+]o-dependent midpoint), where V(the midpoint voltage of the Boltzmann equation) and K00.5 (the Na+ concentration for half-maximal exchange flux activation at Vm = 0) are related by
In terms of the models' rate coefficients, the Na+ concentration for half-maximal activation of Na+/Na+ exchange rate Na/Na at Vm = 0 is given by:
for the pseudo-4-state model (Fig 9 A), or
for the pseudo-2-state model (Fig 9 C), where E is a lumped (apparent) rate constant governing all reaction steps of the exchange cycle except for the external Na+ binding/occlusion and deocclusion/release steps.
Voltage Dependence of Reverse Pumping
A corollary of the compelling evidence, just discussed, for voltage dependence of rate coefficient k-2 is that this same rate coefficient, since it figures in the pseudo-4-state reverse pump reaction scheme of Fig 9 B, may contribute to the voltage dependence of the reverse pumping mode as well. Our findings reported here show that the voltage dependence of reverse pumping is well described by the simplest (i.e., high field access channel) model that accounts for the Na+/Na+ exchange kinetics, with rate expressions entirely analogous to Equation 2a and Equation 2b. In fact (data not shown), we were able to satisfactorily fit simultaneously both Na+/Na+ exchange (data of (fraction of membrane field traversed by external Na+ ions within the access channel), apparent molecularity or Hill coefficient (n) and, hence, apparent valence (z = n
).
If k-2 is, for practical purposes, the sole voltage-sensitive rate coefficient in the pseudo-4-state reverse pumping scheme of Fig 9 B by virtue of a high field access channel effect (Equation 1), then that scheme too can be reduced to a pseudo-2-state model (Fig 9 D). Accordingly, the backward-running pump appears to operate in two steps (a and b). One of these is Na+- and voltage-sensitive because it includes the binding of (at least one) Na+ by a voltage-sensitive mechanism; the other reflects all remaining reactions in the reverse pump cycle. The general expression for the cycling rate, a · b/(a + b), can be cast in two forms (Equation 3aEquation 3b, a and b)
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(3a) |
(a Hill equation with voltage-sensitive apparent K0.5), or
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(3b) |
(a Boltzmann equation with [Na+]o-dependent midpoint), which are identical to those given above (Equation 2aEquation 2b, a and b) for the rate of Na+/Na+ exchange Na/Na except, naturally, for the components of K00.5 for Na+ (pseudo-4-state model; Fig 9 B):
or (pseudo-2-state model; Fig 9 D):
where R is a lumped (apparent) rate constant governing all reaction steps of the backward pumping cycle except for the external Na+ binding/occlusion and deocclusion/release steps. As shown earlier, the characteristic sensitivity of the reverse pump to Vm and [Na+]o (data of Fig 7) is well described by this parsimonious model. The model's merit over more complex ones is its ability to equally account for the kinetics of voltage and Na+ dependence of two modes of operation of the sodium pump (electroneutral Na+/Na+ exchange and reverse pumping) without further assumptions.
Kinetic expressions for more elaborate models of Na+/Na+ exchange also reduce to those derived for the access channel model, provided certain constraints on the individual rate coefficients are satisfied. For example, if both k2 and k-2 in the pseudo-4-state scheme of Fig 8 were voltage-sensitive rate coefficients by virtue of a symmetric Eyring barrier in step 2 (i.e., Na+ deocclusion/release or rebinding/occlusion), the resulting kinetic expressions would be more complex, not only for pump current Ipump in the reverse mode (Fig 9 B), but especially for the flux rate Na/Na in the Na+/Na+ exchange mode (Fig 9 A) because the reciprocating character of the latter causes rate coefficient k2 to govern two of the four steps in the cycle. Specifically, the Ipump-V and
Na/Na -V curves are no longer simple Boltzmann partition functions; the lateral shifts with changes in [Na+]o are not identical for Ipump -V and
Na/Na -V curves, nor are they uniform for each doubling of [Na+]o; the steepness of the curves is not uniform; and the
Na/Na-V curves at various [Na+]o do not tend to the same
Na/Na . Given certain constraints on the relationships between individual rate coefficients, these complex equations for the Eyring barrier model can be made to formally approximate those for the high field access model and satisfy some, but not all, of its criteria. For example, if k-1 << k-2, then each doubling of [Na+]o will cause uniform parallel shifts (i.e., the curves will retain their slopes as they move sideways a fixed distance), but the magnitudes of the lateral shifts will differ between Ipump-V and
Na/Na-V curves, and the slope of the curves will differ for the two modes. We found no model with an Eyring barrier in step 2, whether symmetric (
Comparison of A and B in Fig 9 now raises the question whether the rate coefficient k-3 (Fig 9 B) could have a characteristic voltage dependence sufficiently similar to that of k2 (which occupies an analogous position in the isotope hemicycle of Na+/Na+ exchange, see Fig 9 A) to endow the reverse pumping cycle with a voltage sensitivity formally indistinguishable from that of the Na+/Na+ exchange mode. This possibility is remote for two reasons. First, the voltage sensitivity of the K+ hemicycle is known to be much weaker than that of the Na+ hemicycle (
The average maximum inward pump current we observed in this study was roughly one third to one half as large as the maximum forward Na+/K+ transport current we observed previously in squid giant axon (
Influence of Hypertonic Solutions
Fig 7 shows that hypertonic solutions do not modify the least-squares fit values of the fractional electrical distance () or the apparent molecularity (Hill coefficient n) of our access channel model. However, they do reduce the apparent affinity for Na+
1.5-fold: K00.5 is 715 ± 37 mM in normotonic solutions (Fig 7 A), but 1,065 ± 80 mM in hypertonic solutions (Fig 7 B). This causes a uniform 17-mV leftward shift of the Boltzmann partition curves of Fig 7 B compared with those of Fig 7 A. To examine whether this could be due to additional shielding of negative membrane surface charges by the high ionic strength seawater, we used the
Conclusion
The extracellular Na+ access channel model we proposed for the voltage dependence of the steady-state rate of electroneutral Na+/Na+ exchange (
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Footnotes |
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The present address of Dr. Gadsby is Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021. The present address of Dr. Rakowski is Department of Biological Sciences, Ohio University, Irvine Hall, Athens, OH 45701.
1 Abbreviation used in this paper: H2DTG, dihydrodigitoxigenin.
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Acknowledgements |
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This research was supported by National Institutes of Health grants NS11223 (to P. De Weer), NS22979 (to D.C. Gadsby), and HL36783 (to R.F. Rakowski).
Submitted: 4 August 2000
Revised: 16 January 2001
Accepted: 6 February 2001
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