Correspondence to: Christof Grewer, Max-Planck-Institut für Biophysik, Kennedyallee 70, D-60596 Frankfurt, Germany. Fax:49-69-6303-305 E-mail:grewer{at}mpibp-frankfurt.mpg.de.
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Abstract |
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Electrogenic glutamate transport by the excitatory amino acid carrier 1 (EAAC1) is associated with multiple charge movements across the membrane that take place on time scales ranging from microseconds to milliseconds. The molecular nature of these charge movements is poorly understood at present and, therefore, was studied in this report in detail by using the technique of laser-pulse photolysis of caged glutamate providing a 100-µs time resolution. In the inward transport mode, the deactivation of the transient component of the glutamate-induced coupled transport current exhibits two exponential components. Similar results were obtained when restricting EAAC1 to Na+ translocation steps by removing potassium, thus, demonstrating (1) that substrate translocation of EAAC1 is coupled to inward movement of positive charge and, therefore, electrogenic; and (2) the existence of at least two distinct intermediates in the Na+-binding and glutamate translocation limb of the EAAC1 transport cycle. Together with the determination of the sodium ion concentration and voltage dependence of the two-exponential charge movement and of the steady-state EAAC1 properties, we developed a kinetic model that is based on sequential binding of Na+ and glutamate to their extracellular binding sites on EAAC1 explaining our results. In this model, at least one Na+ ion and thereafter glutamate rapidly bind to the transporter initiating a slower, electroneutral structural change that makes EAAC1 competent for further, voltage-dependent binding of additional sodium ion(s). Once the fully loaded EAAC1 complex is formed, it can undergo a much slower, electrogenic translocation reaction to expose the substrate and ion binding sites to the cytoplasm.
Key Words: glutamate transporter, charge movement, patch clamp, caged compounds, rapid kinetics
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INTRODUCTION |
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In the mammalian brain L-glutamate is the major excitatory neurotransmitter (
According to the proposed stoichiometry, a total of two positive charges is moved in the inward direction during a single turnover of the glutamate transporter. Thus, glutamate transport is electrogenic and induces transmembrane currents. At present, only one electrogenic partial reaction of the transporter has been identified that is responsible for <20% of the total charge movement, namely the binding of sodium ions on the extracellular side or conformational changes of the transport protein linked to it (-aminobutyric acid (GABA)1 transporter (
Closely linked to this issue are questions about the sequence and mechanism of Na+ and glutamate binding on the extracellular side of the transporter. In general, such questions can be answered by investigating the dependence of transport currents on the concentration of cotransported ions, as we have recently demonstrated by determining the binding sequence of protons and glutamate to the EAAC1 (
To elucidate the transporter mechanism in more detail, we determined in this work the influence of the extracellular sodium ion concentration and the transmembrane potential on the steady-state and on the presteady-state currents of a neuronal subtype of the glutamate transporter excitatory amino acid carrier 1 (EAAC1) with a time resolution of 100 µs. This time resolution was achieved by applying the method of laser-pulse photolysis of caged glutamate that we introduced recently for studying rapid glutamate transporter reactions (
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MATERIALS AND METHODS |
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Expression of EAAC1 in Mammalian Cells
Rat EAAC1 cloned from rat retina (
Electrophysiology
Glutamate-induced EAAC1 currents were recorded with an amplifier (model EPC7; Adams and List) under voltage-clamp conditions in the whole-cell current-recording configuration (. EAAC1-mediated uncoupled anion currents can be increased by using the more permeant anion SCN- instead of chloride (
Laser-pulse Photolysis and Rapid Solution Exchange
The rapid solution exchange (time resolution 100200 ms) was performed by means of a quartz tube (opening 350 µm) positioned at a distance of
0.5 mm to the cell. The linear flow rate of the solutions emerging from the opening of the tube was
510 cm/s. Laser-pulse photolysis experiments were performed as described previously (
CNB-caged glutamate (Molecular Probes;
Data were recorded using the pClamp6 software (Axon Instruments), digitized with a sampling rate of 1 kHz (solution exchange) or 25 kHz (laser photolysis and Na+ binding transient currents,) and low-pass filtered at 250 Hz or 310 kHz, respectively.
Data Evaluation and Terminology
For simplicity, the following terminology was used. The glutamate-induced coupled transport current was termed IGlu-Na+/K+ in the inward transport mode and IGlu-Na+ in the Na+/glutamate homoexchange mode. The uncoupled anion current was named IGlu-anionic for the glutamate-dependent component and Ianionic for the glutamate-independent component.
Nonlinear regression fits of experimental data were performed with Origin (Microcal Software) or Clampfit (pClamp8 software; Axon Instruments) by the use of the following equations. The presteady-state currents of the anionic current (IGlu-anionic, in the presence of SCN-) were fitted with a sum of two exponential functions and a steady-state current component (Iss): I = I1 · exp(-t/rise) + I2 · exp(-t/
decay) + Iss. The presteady-state transport currents IGlu-Na+/K+ and IGlu-Na+ (in the absence of SCN-) were fitted with a sum of three exponential functions and a stationary current component: I = I1 · exp(-t/
rise) + I2 · exp(-t/
decay1) + I3 · exp(-t/
decay2) + Iss. Under homoexchange conditions, Iss became zero. The observed time constants of
rise of IGlu-anionic were in the range of
1 ms and, therefore, were similar to the time constants of
decay1 of IGlu-Na+/K+ and IGlu-Na+. For this reason, we named these time constants in the remainder of this article
fast. A similar time dependence with
8 ms was found for the time constants
decay of IGlu-anionic, and
decay2 of IGlu-Na+/K+ and IGlu-Na+. These time constants were named
slow.
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RESULTS |
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The time course of presteady-state kinetics of EAAC1 currents recorded in the anion-conducting mode (IGlu-anionic) after a glutamate concentration jump induced by laser-pulse photolysis of caged glutamate shows a two-exponential behavior (50 ms. The kinetics are different from those observed for IGlu-anionic in two ways. First, the maximum of the current is shifted to shorter times; and, second, the decay of the current occurs with two exponentials. Attempts to fit the decay of IGlu-Na+/K+ with only one exponential component yielded significantly inferior fits compared with those with two exponential components, as shown by the analysis of the fitting residuals in Fig 1 B (top panel). We determined a time constant of
= 0.9 ± 0.1 ms (n = 6) for the rapidly decaying phase of IGlu-Na+/K+, in the same range as
= 0.8 ± 0.1 ms (n = 20) for the rising phase of the anionic component of EAAC1 currents (
= 8.8 ± 1.1 ms (n = 6), which is similar to the time constant we found for the decay of IGlu-anionic (
= 8.7 ± 0.4 ms, n = 20), again suggesting that they represent the same kinetic process. For simplicity, we named the time constants according to their magnitude
fast and
slow (see MATERIALS AND METHODS).
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Presteady-state Currents in the Na+/Glutamate Homoexchange Mode
So far, we determined the presteady-state kinetics of EAAC1 in the inward transport mode. We then asked the question whether the same charge movements are present when the amino acid substrate and Na+ are applied to either side of the membrane and potassium ions are absent (homoexchange mode). Under these conditions, reactions related to the relocation of the K+-bound transporter are eliminated, thus, allowing one to isolate the Na+/glutamate half cycle of EAAC1. The result of this experiment is shown in Fig 1 C. The homoexchange current, IGlu-Na+, has a very similar time dependence as IGlu-Na+/K+ in the inward transport mode. However, as expected for electroneutral equilibrium exchange, there is no steady-state current component observed, within experimental error. In analogy to IGlu-Na+/K+ in the inward transport mode, the decay of the transient current is a double exponential process, with a rapidly and a slowly decaying component. As in the inward transport mode, the rate constant of the decay of the rapid phase of IGlu-Na+ is similar to the rise of the anionic current component in the presence of SCN- (IGlu-anionic), as illustrated in Fig 1 D where both currents were corrected for the contribution of the slow phase and scaled to the same amplitude. These results demonstrate that both, the fast and the slow charge movements are not associated with relocation steps of the K+-bound transporter, but rather with the Na+/glutamate translocation limb of the transport cycle.
To further characterize the two kinetic phases in the Na+/glutamate translocation limb of the transport cycle, we determined their voltage dependence.
Voltage Dependence of Presteady-state EAAC1 Currents
Typical glutamate-induced fast EAAC1 currents in the homoexchange mode (IGlu-Na+) as a function of the transmembrane potential (Vm) are shown in Fig 2 A. Clearly, the transient IGlu-Na+ component increases with decreasing transmembrane potential (Fig 2 A), is always inwardly directed, and has a tendency to saturate at strongly negative Vm. The rate constant of the decay of the slow current component (1/slow of IGlu-Na+) increases exponentially with decreasing Vm (Fig 2 B, open circles). This voltage dependence of 1/
slow is similar in the inward transport mode (IGlu-Na+/K+, closed circles). In addition, 1/
slow for the anionic current component IGlu-anionic in the inward transport mode is shown (open triangles), demonstrating the same voltage dependence, within experimental error. The qualitatively identical voltage dependence of
slow determined from IGlu-Na+/K+ and from IGlu-anionic show that they are associated to the same kinetic process in the Na+/glutamate translocation limb of the transport cycle. These results further indicate that EAAC1 charge movements are strongly inwardly driven, even in the equilibrium exchange mode. Linear regression of the log(1/
slow) versus Vm relationship yields a slope of (-4.7 ± 0.2) x 10-3/mV (Fig 2 B, solid line). This corresponds to a fraction of 0.56 of the transmembrane electric field sensed by this process if one charge was moved.
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In principle, the voltage dependence of relaxation rate constants and charge movements could be influenced by the distribution of the initial states of the carrier before the glutamate concentration jump. However, in the absence of extracellular K+ and glutamate, it is reasonable to assume that EAAC1 is locked in a state (or states) with the glutamate binding site exposed to the external side of the membrane.
In contrast to 1/slow, the rate constant for the fast decaying phase of IGlu-Na+/K+ was only slightly affected by the voltage and increased with increasing the potential (Fig 2 B, closed circles). The same voltage dependence was found for the time constant of the rising phase of IGlu-anionic (
fast see MATERIALS AND METHODS) as shown in Fig 2 B (open triangles), indicating that both rate constants reflect the same reaction process of EAAC1. Comparison of the voltage dependence of
slow and
fast further show that the slope of (8.8 ± 2.2) x 10-4/mV of the log(1/
fast) versus Vm relationship is only about one fifth of the slope found for the voltage dependence of log(1/
slow) and has the opposite sign. Together, these results suggest that the rapidly decaying current component of IGlu-Na+/K+ and IGlu-Na+ (or rapidly rising current component of IGlu-anionic) is associated with an inward charge movement, however, its rate of decay (or rise) is determined by a process that is only weakly and oppositely dependent on the transmembrane potential compared with the slow phase.
In the following paragraphs, we describe the more detailed characterization of these charge movements and their relationship to the binding of extracellular sodium ions and the translocation of substrate across the membrane.
Glutamate-independent Fast Charge Movements Are Inhibited by TBOA
Wadiche and co-workers (
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In line with results obtained by Wadiche and co-workers ( for EAAC1 in HEK293 cells was voltage independent and at least 20-fold faster with an average time constant of 150 ± 30 µs (n = 12, 2 cells). The voltage independence implies that the determination of this time constant is most likely limited by the time resolution of the voltage clamp (estimated to
100 µs from typical series resistance and cell capacitance values, the filter frequency was 3 kHz). Therefore, even though the charge movements are detectable, their time course is distorted by the rate-limiting voltage clamp. However, the existence of these TBOA-sensitive transient charge movements indicate that EAAC1, in analogy to EAAT2, binds sodium ions in a very rapid process before the glutamate binding process occurs (see next paragraphs). For EAAT2, it was found that the inhibitor-sensitive transient charge movements are abolished in the absence of extracellular Na+ (
Na+ Concentration Dependence of the Leak Anion Conductance
As shown by others (2 twice as high compared with the case of n = 1 (Fig 3 C). However, within experimental error, it is impossible to differentiate if binding of one or two Na+ ions is associated with this process.
Steady-state Currents: [Na+] Dependence of KM
To characterize the [Na+] dependence of EAAC1 currents in more detail, we performed experiments under conditions of steady-state inward transport (IGlu-anionic). First, we investigated changes in affinity of EAAC1 for glutamate depending on the extracellular sodium ion concentration. Glutamate in different concentrations was applied to voltage-clamped HEKEAAC1 cells (Vm = 0 mV) with a rapid solution exchange device. In the whole Na+ concentration range (5140 mM) studied, the glutamate dependencies could be well fitted by Michaelis-Mentenlike relationships (Fig 4 A). From the fits, we determined the apparent glutamate dissociation constants and the current at saturating glutamate concentrations Imax. As seen in Fig 4 A, reduced extracellular Na+ concentrations lead to a pronounced increase of the apparent KM for glutamate from 7.2 ± 1.1 µM at 140 mM Na+ to 380 ± 50 µM at 5 mM external Na+. The [Na+] dependence of KM is shown in a logarithmic plot in Fig 4 B.
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[Na+] Dependence of the Glutamate-induced Steady-state Current Imax
By comparing the maximum currents (Imax) induced by saturating glutamate concentrations at different extracellular Na+ concentrations, one can investigate the binding order of Na+ with respect to glutamate. The equations for the different models are shown in Table 1. As demonstrated in Fig 4 C, Imax shows a strong [Na+] dependence; lowering the external Na+ concentration leads to a significant decrease in the maximum current. If all three sodium ions bind rapidly to the transporter before the glutamate binding process takes place, Imax should be the same in the whole Na+ concentration range (see Table 1 and DISCUSSION). Thus, we can exclude this model. Therefore, the results suggest a difference in the binding sequence of protons and Na+ with respect to glutamate. Whereas protons can bind to the empty transporter before glutamate, at least one Na+ must bind to the substrate-loaded carrier. The fits shown in Fig 4 C represent calculations according to the Hill equations with Hill coefficients of either one or two, indicating that one or two sodium ions bind after glutamate.
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Na+ Concentration Dependence of IGlu-anionic Presteady-state Kinetics
As shown in Fig 1, the fast and the slow charge movements are observed for the coupled transport current IGlu-Na+/K+ as well as for homoexchange (IGlu-Na+), indicating that the inwardly directed transient current is not influenced by the intracellular sodium ion concentration. Thus, to test if the presteady-state current is associated with binding of extracellular Na+, we determined its Na+ concentration dependence. Since lowering the extracellular sodium ion concentration leads to a drastic reduction of the IGlu-Na+/K+ current component, the first measurements were done instead with IGlu-anionic, which also contains information about fast and
slow. In Fig 5 A, photolysis experiments at different extracellular Na+ concentrations ([Na+]o) are shown. At 140 mM extracellular Na+, photolytic release of saturating concentrations of glutamate from
CNB-caged glutamate leads to a rise of the current to a maximum (
fast = 0.8 ± 0.1 ms, n = 3) and a decay of the transient current to a steady state (
slow = 8.7 ± 0.4 ms, n = 3) that is consistent with previous reports (
slow showed a strong [Na+] dependence (Fig 5 B, top panel). It increases from 14.3 ± 1.4 ms at 49 mM [Na+]o, to 19.9 ± 2.9 ms at 18 mM [Na+]o, to 33.1 ± 5.2 ms at an extracellular Na+ concentration of 10 mM. In contrast, the fast time constant for the rising phase of the transient current (
fast) is almost [Na+] independent as seen in Fig 5 B (top panel; see Appendix, Equation 1). This is consistent with the results obtained for the time constant of the fast decaying component of IGlu-Na+/K+, which is also essentially [Na+] independent (Fig 5 B), further supporting the view that these processes are associated with the same reaction of EAAC1.
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The Magnitude of the Fast Charge Movement Is Na+ Concentration-dependent
To confirm that the Na+-binding reaction preceding the glutamate binding step does not cause the fast charge movements, as suggested above, we performed laser-pulse photolysis experiments detecting IGlu-Na+/K+ at low extracellular Na+ concentrations. In case of a voltage-dependent Na+ preequilibrium preceding glutamate binding, the charge movements should increase with decreasing Na+ concentration. The opposite is found experimentally, as demonstrated in Fig 6 A. The left panel shows the current (IGlu-Na+/K+) recorded in a typical laser-pulse photolysis experiment (1 mM caged glutamate) in the presence of 140 mM extracellular Na+. To ensure that the [Na+]o effect is not only caused by a change in the apparent KM of EAAC1 for glutamate (see previous paragraphs) we raised the glutamate concentration by increasing the concentration of caged glutamate fourfold to 4 mM in the experiments with 20 mM Na+. The total charge obtained by integrating the fast component of the current at 140 mM Na+ is 47 fC. After decreasing the external Na+ concentration to 20 mM, the amplitude of the transient current is reduced by a factor of 3.7 (Fig 6 A, right trace). The charge of the fast component is 15 fC. In four experiments with two cells, we obtained an average charge ratio Q(20 mM Na+)/Q(140 mM Na+) of 0.39 ± 0.06 (Fig 6 B). The time constant for the decay of the fast current component fast was 0.9 ± 0.2 ms (n = 6, 3 cells, 20 mM Na+) and, therefore, unchanged when compared with the results obtained at 140 mM extracellular Na+.
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The voltage dependence of the charge carried by the rapid process is shown in Fig 6 C for IGlu-Na+ in the presence of 140, 20, or 10 mM Na+. At high sodium ion concentrations, the moved charge Q integrating the fast current component is invariant at Vm more negative than about -30 mV (Qmax = 620 ± 25 fC) and slightly decreases to 240 ± 28 fC (n = 8, 3 cells) at Vm = +75 mV. When fit to a Boltzmann-like relationship (see Appendix, Equation 2) a midpoint potential (VQ) of 53 ± 7 mV and a apparent valence of 0.61 ± 0.08 are obtained (see Appendix, Equation 3 and Equation 4). At low Na+ concentrations (10 and 20 mM), in general, Q demonstrates the same dependence on the transmembrane potential; however, the midpoint potential of the charge movement is shifted to -48 ± 5 mV (10 mM Na+) and -21 ± 4 mV (20 mM Na+) with an apparent valence of 0.55 ± 0.05, as expected for a shift of the voltage-dependent Na+-binding equilibrium to the less-occupied form at low Na+ concentrations, according to Equation 3 (see Appendix). From the shift in the midpoint potential, which is shown in Fig 6 C (inset), the apparent valence is calculated as 0.65, in agreement with the slope factor of the Boltzmann relationships at each Na+ concentration. This finding is in agreement with a single voltage-dependent Na+-binding reaction of the EAAC1Na+glutamate complex. It should be noted that at Na+ concentrations of 10 and 20 mM, saturation of the charge movements at very negative potentials was not achieved due to the limited accessible range of transmembrane potentials. Therefore, the stated VQ values at these Na+ concentrations represent lower estimates of the true VQ.
In addition, we determined the charge moved during the slow process that is shown in Fig 6 D. This charge movement is independent of the transmembrane potential, within experimental error (n = 8, 3 cells). Both charge movements are under saturating conditions of similar magnitude (Fig 6C and Fig D). Together, the results imply that charge transfer depends on extracellular Na+ and the transmembrane potential, but the rate of the deactivation process of the current does not. Such a Na+ concentration dependence can be explained with a two-step mechanism in which a slow and Na+- and voltage-independent reaction governs the rate of the current decay, whereas a subsequent rapid and electrogenic Na+-binding step is responsible for the inward charge movement.
Voltage Dependence of EAAC1 at Reduced External [Na+]
It was previously suggested that sodium ion binding may become rate limiting for the glutamate transporter turnover at low extracellular Na+ concentrations (slow is essentially unchanged. The slope of the log(1/
slow) versus Vm relationship at 10 mM Na+ is (-5.6 ± 0.1) x 10-3/mV, which is very close to that obtained at a Na+ concentration of 140 mM ((-5.5 ± 0.3)x 10-3/mV). Consistent with the results obtained at 140 mM extracellular Na+, the rate of the current rise is almost voltage independent.
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DISCUSSION |
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Here, we have identified short-lived intermediates of the Na+-binding and glutamate translocation reaction of EAAC1 by using a rapid chemical-kinetic technique that we introduced recently to study glutamate transporter function (
The Nature of the Presteady-state Charge Movements
The main characteristic of the presteady-state charge movement is that its decay is two-exponential. Thus, the results directly point to the existence of two individual electrogenic reactions that are separated on the time scale. These reactions are also observed in the total absence of intracellular and extracellular K+, confirming our previous suggestion that they are related to the translocation of the Na+ and glutamate-loaded carrier, but not the relocation of the K+-bound form of EAAC1 (
Which glutamate transporter reactions steps contribute to the inward presteady-state IGlu-Na+/K+ current? It has been previously shown that Na+ can bind to another transporter subtype (EAAT2) in the absence of glutamate (
A General Model for the Na+/Glutamate Translocating Half Cycle of EAAC1
The kinetic model that we propose for the Na+/glutamate translocating half cycle of EAAC1 is shown in Fig 8 A and is based on sequential, but not random, binding of sodium ions and glutamate to their extracellular binding sites on the transporter protein. In this model, glutamate binds to the Na+EAAC1 complex, and bound glutamate is absolutely necessary for the binding of further sodium ion(s) to EAAC1. In fact, we propose a structural transition of EAAC1 occurring on a millisecond-to-submillisecond time scale that is induced by glutamate binding and renders the transporter competent for binding of one or two more sodium ions. We include such a structural transition in the model based on the following experimental findings that are otherwise difficult to reconcile. First, the fast glutamate-induced charge movement increases with increasing Na+ concentration (at saturating [glutamate]), but its rate of decay does not. Second, the fast glutamate-induced charge movement increases with decreasing membrane potential, however, its rate of decay is essentially Vm independent. Third, it is unaffected by intracellular K+, Na+ and glutamate and, thus, not due to dissociation reactions on the cytoplasmic face of EAAC1. Fourth, the maximum anionic current increases with increasing Na+ concentration, but its rate of formation is Na+ independent; and fifth, the rate of formation of IGlu-anionic is essentially voltage independent. Together, these observations suggest, independent of a kinetic model, the existence of an electroneutral transporter reaction step that determines the rate of decay of the charge movement and that is unaffected by extracellular Na+ and by the transmembrane potential. It is followed by a Na+-dependent and electrogenic reaction that is in rapid preequilibrium with respect to the rate-limiting step. This latter step, which we interpret as a voltage-dependent Na+ binding reaction (Fig 8 A) is responsible for the fast phase of the glutamate-induced charge movement that we observe.
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Previously, we assigned the slow phase of the glutamate-induced charge movement to the Na+/glutamate translocation step across the membrane (
The general model shown in Fig 8 A can explain all of the results obtained in this study qualitatively. To demonstrate this, we performed analytical calculations based on the equations listed in Table 1 and numerical simulations of the time dependence of transport currents based on Equation 5 (see Appendix). Some of the results of these simulations are shown in Fig 9 and demonstrate the general good agreement between the experimental data and the predictions of the proposed mechanism regarding the time dependence of transport currents and the effect of the extracellular Na+ concentration on the currents.
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Are the Rapid Charge Movements Gating Currents?
Gating currents are generally observed in voltage-gated ion channels (
Previously, it was speculated that glutamate itself, when bound to the transporter, provides the gating particle and part of the permeation pathway for anions, possibly by contributing its positively charged -amino group (
EAAC1 Properties at Steady-state
The major results from the Na+ concentration dependence under conditions of steady-state transport are the following: (1) the apparent affinity of EAAC1 for glutamate (KM) decreases markedly with decreasing concentration of extracellular Na+; and (2) the maximum glutamate-induced current (Imax) decreases with decreasing [Na+]. We have derived expressions to quantitatively describe the Na+ concentration dependence of KM and Imax for a number of different models as shown in Table 1. The expressions are based on the known stoichiometry of EAAC1 (three cotransported Na+ ions per glutamate;
Stoichiometry of Na+ Binding
Although the data presented here strongly support the sequential Na+ and glutamate binding model shown in Fig 8 A, they do not allow us to differentiate between the models NaTSNa2 and Na2TSNa. The voltage dependence of the final Na+-binding step is best approximated with a model that involves only one Na+ ion (Na2TSNa). In contrast, the inhibition of the leak anion current by TBOA, which is a measure of the initial Na+-binding reaction, is best fit with an apparent Hill coefficient of 1, thus, supporting model NaTSNa2. The effect of the extracellular Na+ concentration on Imax, Km for glutamate, and the rate constants determined from the presteady-state experiments can be equally well described by both models (Fig 9). Therefore, we conclude that, based on these data, either one of the two Na+-binding stoichiometries would be possible. The clarification of this issue will need further, more detailed experiments.
Comparison to Previous Studies
First, our results confirm previous proposals that positive charge is transferred in the Na+/glutamate translocation step of the transporter (
Second, we present evidence that the glutamate translocation reaction is strongly inwardly driven at negative transmembrane potentials close to the physiologically important range. This result is in good agreement with previous studies on the deactivation of EAAT2 anion currents in the homoexchange mode upon rapid removal of glutamate (
Third, we confirmed previous results suggesting that initial binding of sodium ions to the transporter senses part of the transmembrane electric field (
Finally, it is interesting to note that conformational changes induced by binding of glutamate to the glutamate transporter subtype Glt-1 have been proposed to explain glutamate-induced accessibility changes of specifically introduced cysteine residues that are believed to be not directly part of the glutamate binding site to sulfhydryl-reactive reagents (
Comparison to Other Na+-coupled Transporters
In general, it is believed that association of Na+ with a binding site localized within the transmembrane electric field or conformational changes linked to it is a major source of the electrogenicity of Na+-driven transport systems. Our results obtained with EAAC1 confirm such an interpretation. In EAAC1, binding of at least two sodium ions on the extracellular side contribute to the overall charge movement, even though to a relatively minor extent. For many other transporters, however, the substrate translocation steps that follow extracellular substrate and Na+-binding are thought to be electroneutral. Such electroneutral behavior was found for the Na+ dependent transporters for glucose (
Another general feature of Na+-coupled carriers is that their function is based on a charge balance mechanism. In the sodium glucose transporter, the positive charge of the two cotransported sodium ions is compensated by two negative charges on the transporter that move together with the organic substrate in the electric field (
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Footnotes |
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1 Abbreviations used in this paper: CNB,
-carboxy-2-nitrobenzyl; EAAT, excitatory amino acid transporter; EAAC1, excitatory amino acid carrier 1; GABA,
-aminobutyric acid; Glt-1, glutamate transporter 1; HEK, human embryonic kidney; TBOA, DL-threo-ß-benzyloxyaspartatic acid.
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Acknowledgements |
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We thank T. Rauen for providing EAAC1 cDNA and helpful discussions, and K. Hartung for critical reading of the manuscript. We thank K. Shimamoto for providing TBOA.
This work was supported by the Deutsche Forschungsgemeinschaft (grant No. GR 1393/2-1 awarded to C. Grewer).
Submitted: 12 February 2001
Revised: 13 April 2001
Accepted: 16 April 2001
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Appendix |
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When concentrations of glutamate and Na+ are used that saturate their initial binding sites on EAAC1 the model shown in Fig 8 A can be reduced to the following pseudotwo-state model Scheme 1:
Here, the binding of the final Na+ to EAAC1 is thought to be in rapid equilibrium compared with the transition NaTS NaT'S. Thus, the NaT'S and the NaT'SNa states can be lumped into one state. According to this pseudotwo-state model, the observed relaxation rate constant for the rapid decay of IGlu-Na+/K+, 1/
fast, can be expressed as:
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(1) |
Here, k1 and k-1 are the rate constants for the transitions NaTS NaT'S and NaT'S
NaTS, respectively, and KNa+2/3 is the dissociation constant of the sodium ion from its site on NaT'S. The following assumptions were made: (1) the glutamate concentration is saturating for the initial Na+ binding step; (2) binding of glutamate and of the initial Na+ ion are in rapid preequilibrium with respect to the transition NaTS
NaT'S. Thus, k1 becomes glutamate and Na+ independent; and (3) glutamate translocation is comparatively slow and, therefore, can be neglected.
Using the same assumptions, the charge moved during the rapid phase of the current decay can be calculated as:
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(2) |
In this equation, Qmax is a scaling factor and represents the maximum amount of charge movement, which depends on the number of EAAC1 molecules under observation, the elementary charge and the apparent valence of this charge, zQ. Additionally, it was assumed that the transporter resides fully in the state NaTS until the reaction is initiated. When evaluated in a voltage-dependent manner, Equation 2 resembles a Boltzmann-like relationship. The midpoint potential of the charge movement, VQ, is [Na+] dependent and can be given as:
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(3) |
with R being the gas constant, T being the temperature, and F being the Faraday constant. The voltage dependence of the charge movement is introduced by the voltage dependence of KNa+2/3(Vm) = k-Na/k+Na, which can be expressed as follows, according to transition-state theory (
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(4) |
where KNa+2/3(0) represents the dissociation constant of Na+ from EAAC1 at Vm = 0 mV.
For the simulations of the time dependence of transport currents the following equation was used:
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(5) |
Here, P denotes the fractional occupancy of the respective state as defined in the model in Fig 8 A. T0 is the number of transporters under observation, e is the elementary charge, and k+Na and k-Na are the rate constants for binding of the final Na+ to EAAC1 and dissociation of this Na+ from EAAC1, respectively. The time dependence of the current can be obtained by calculating the time dependencies of the fractional state populations P by numeric integration of the differential equations pertaining to the kinetic scheme shown in Fig 8 A. For simplicity, n (the number of initial Na+ binding reactions) was set to 2. Thus, the binding of Na+ to the glutamateEAAC1 complex involves only one Na+ ion.
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