Correspondence to: Christof Grewer, Max-Planck-Institut für Biophysik, Kennedyallee 70, D-60596 Frankfurt, Germany. Fax:49-69-6303-305 E-mail:christof.grewer{at}mpibp-frankfurt.mpg.de.
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Abstract |
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Uptake of glutamate from the synaptic cleft is mediated by high affinity transporters and is driven by Na+, K+, and H+ concentration gradients across the membrane. Here, we characterize the molecular mechanism of the intracellular pH change associated with glutamate transport by combining current recordings from excitatory amino acid carrier 1 (EAAC1)expressing HEK293 cells with a rapid kinetic technique with a 100-µs time resolution. Under conditions of steady state transport, the affinity of EAAC1 for glutamate in both the forward and reverse modes is strongly dependent on the pH on the cis-side of the membrane, whereas the currents at saturating glutamate concentrations are hardly affected by the pH. Consistent with this, the kinetics of the presteady state currents, measured after saturating glutamate concentration jumps, are not a function of the pH. In addition, we determined the deuterium isotope effect on EAAC1 kinetics, which is in agreement with proton cotransport but not OH- countertransport. The results can be quantitatively explained with an ordered binding model that includes a rapid proton binding step to the empty transporter followed by glutamate binding and translocation of the proton-glutamate-transporter complex. The apparent pK of the extracellular proton binding site is 8. This value is shifted to
6.5 when the substrate binding site is exposed to the cytoplasm.
Key Words: glutamate transporter, patch-clamp, laser-pulse photolysis, rapid kinetics, reverse transport
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INTRODUCTION |
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L-Glutamate is the major excitatory neurotransmitter in the mammalian brain (
Additionally, movement of a pH-changing ion is associated with glutamate transport because glutamate uptake mediates intracellular acidification (-carboxylate group induced by a major pK change of this group after binding of the negatively charged substrate to the transporter (
Recently, we introduced the method of laser-pulse photolysis of caged glutamate to investigate the presteady state kinetics of glutamate transporters with a time resolution in the 100-µs range (
The results demonstrate that EAAC1 has to be protonated before glutamate binds at the extracellular side and charge translocation takes place. This implies the existence of an ionizable amino acid residue in the protein with an apparent pK of 8 that is responsible for proton cotransport. The dissociation of glutamate on the intracellular side of the transporter is controlled by a pK shift of this residue by at least 1.5 pK units that takes place after glutamate translocation.
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MATERIALS AND METHODS |
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Expression of EAAC1 in Mammalian Cells
EAAC1 cloned from rat retina (
Electrophysiology
Glutamate-induced EAAC1 currents were recorded with an amplifier (model EPC7; Adams & List) under voltage-clamp conditions either in the whole-cell current-recording configuration or in the inside-out patch-clamp configuration ( (whole-cells) or 300400 k
(inside-out giant patches). EAAC1-associated currents are composed of two components, a coupled transport current and an uncoupled current carried by anions termed IGlu-Na+/K+ and IGlu-anionic, respectively (
5 mM) were used, the nonactivating bath solution contained additional amounts of Tris (up to 8 mM) to balance the osmolarity. The same solutions were used for the reverse transport studies. To avoid the formation of vesicles in the inside-out patch configuration, the pipette solution contained no CaCl2 (
Laser-pulse Photolysis and Rapid Solution Exchange
The rapid solution exchange (time resolution 100 ms) was performed with a quartz tube (inner diameter 350 µm) positioned 0.5 mm from 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 (
-Carbonyl-2-nitrobenzyl (
CNB)caged glutamate (Molecular Probes) in concentrations of 1 mM (pH 6.0 and 7.4) to 4 mM (pH 9.0) or free glutamate was applied to the cells, and photolysis of the caged glutamate was initiated with a light flash (340-nm, 15-ns excimer laser pumped dye laser; Lambda Physik). The light was coupled into a quartz fiber (diameter, 365 µm) that was positioned in front of the cell in a distance of 300 µm. With maximum light intensities of 500600 mJ/cm2, saturating glutamate concentrations could be released, which were tested by comparing the photolysis-induced steady state current with that generated by rapid perfusion of the same cell with 100 µM glutamate (pH 6.0 and 7.4) or 500 µM glutamate, pH 9.0 (
Data were recorded using the pClamp6 software (Axon Instruments), digitized with a sampling rate of 1 kHz (solution exchange) or 25 kHz (laser-pulse photolysis) and low passfiltered at 250 Hz or 3 kHz, respectively. Nonlinear regression fits of experimental data were performed with Origin (Microcal software) or Clampfit (pClamp8 software; Axon Instruments).
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RESULTS |
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The basic experiments on the effect of pH on EAAC1 expressed in HEK293 cells are shown in Fig 1 D (right), demonstrating results that differ from those reported by
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pH Dependence of the KM for Glutamate
In the following experiments the highly permeant anion SCN- was used as the main intracellular anion to increase transporter-associated currents (
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Subsequently, the pH dependence was examined in the reverse transport mode under conditions of steady state transport by using the patch-clamp technique in the inside-out configuration (30% of the glutamate is protonated (pK 4.4), the affinity for glutamate once again decreases dramatically and reaches a KM of 820 µM (Table 1). To exclude that this effect is due to the increased ionic strength imposed by the succinate-Tris buffer, the apparent KM for glutamate at pH 7.4 in the forward transport mode was determined in the presence of succinate-Tris buffer, showing no difference to the results obtained with a Mes-Tris buffer.
It has been reported previously that the apparent KM of glial transporter subtypes for glutamate at pH 7.3 does not change depending on whether the uncoupled anion current(IGlu-anionic) or exclusively the coupled transport current (IGlu-Na+/K+) is measured (
pH Dependence of the Glutamate-induced Current Imax
To investigate the binding sequence of the proton and glutamate to the transporter, the maximum currents (Imax) at saturating glutamate concentrations were compared in a pH range between 6.0 and 10.0. These experiments were performed by measuring IGlu-Na+/K+ in the whole-cell current recording configuration of voltage-clamped HEKEAAC1 at 0 mV. As shown in Fig 1 D (left), Imax does not depend significantly on the proton concentration under these conditions. This kind of analysis allows the direct experimental determination of the protonglutamate binding sequence: the fact that Imax is not affected by the pH indicates that proton binding occurs first, followed by glutamate binding. The pH effect on IGlu-anionic was also determined. In contrast to IGlu-Na+/K+, the glutamate-induced anionic currents exhibit a pH dependence, showing a moderate increase of IGlu-anionic with decreasing pH (Fig 1 D, middle).
Finally, to test if the pH dependence is qualitatively similar in the reverse transport mode, the same experiments were performed using inside-out patches excised from HEKEAAC1. Consistent with the results obtained in the forward transport mode, a slight pH dependence was also observed for IGlu-anionic. Compared with the current at pH 8.0, which was induced by saturating glutamate concentrations, Imax for pH 7.4 and 6.0 was 1.13 ± 0.04 (n = 3) and 1.27 ± 0.03 (n = 3), respectively.
Effect of Proton Concentration on Presteady State Kinetics of EAAC1
The laser-pulse photolysis method of caged glutamate was used to determine the pH effect on the presteady state kinetics of EAAC1 and resolved a rapid transient current component preceding the steady state current in the presence of thiocyanate in the pipette (1 mM caged glutamate, 125 µM released glutamate; Fig 2 A, middle trace). The transient current results from the rapid synchronized formation of a glutamate-gated anion-conducting state that is followed by the subsequent population of other transporter states (desynchronization of the transporters) as it approaches a new steady state (
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Assuming that the glutamate binding follows the proton binding step and proton binding is fast, the presteady state kinetics of EAAC1 should not be affected by pH changes at saturating glutamate concentrations. In contrast, if glutamate binds first to the transporter and the proton binding step follows subsequently, the rate of formation of the proton-glutamate-transporter complex should depend on the proton concentration. Furthermore, at low proton concentrations, it is expected that this would slow down the rise and decay time for the transient current, even if the glutamate concentration is saturating. To differentiate between these two possibilities, the presteady state currents upon photolytic release of saturating glutamate concentrations were monitored additionally at pH 6.0 and 9.0 (500 µM released glutamate) as shown in Fig 2 A. Despite the small change in the current amplitude, which was already observed under conditions of steady state transport, the presteady state kinetics of EAAC1 are not substantially altered and are not pH-dependent. Consistent with this, the time constants for the formation and the decay of the transient current component are not significantly influenced by the pH (Fig 2 B), indicating that the pH specifically affects EAAC1 proton transport, but not the kinetics and function of EAAC1 by unspecific effects, within the pH range examined. Photolytic release of subsaturating concentrations of glutamate at pH 9.0, however, leads to reduced rates for the formation and deactivation of the transient current (not shown). This effect is indistinguishable from that observed at pH 7.4, but its dose dependence is shifted to higher glutamate concentrations.
To test if the photolysis rate of caged glutamate, which is known to be pH-dependent from analogous compounds (CNB-caged compound photolysis reaction, the aci-nitro intermediate, has a characteristic absorption at 430 nm and its decay kinetics are commonly accepted to represent the release kinetics of the caged substrate (
40 times faster than the rise of the whole-cell current in the transient kinetic experiment (Fig 2 A). In addition, the photolysis quantum yield (
) was measured as a function of the pH, as shown in Fig 3 B. The quantum yield is slightly pH-dependent, exhibiting a maximum between pH 7 and 8. At pH 9.0 (
= 0.1, n = 3) it has 71% of its value at pH 7.0 (
= 0.14;
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pH Effect on CurrentVoltage Relationships of EAAC1
So far, the data were collected at 0 mV transmembrane potential and they are consistent with an ordered binding sequence of the proton, followed by glutamate to the transporter. Furthermore, the data indicate that even at a proton concentration of 0.1 nM, pH 10, protonation is not rate limiting for the overall turnover of the transporter, as demonstrated by the kinetic experiments. To test if this is also correct at different transmembrane potentials, the influence of the pH on the voltage dependence of steady state EAAC1 currents was determined. The voltage dependence of the steady state IGlu-Na+/K+ current (symmetrical Cl- on both sides of the membrane) at pH 7.4 is shown in Fig 4 A. Consistent with previous results, this current is inwardly directed, increases with decreasing transmembrane potential, and does not reverse within the voltage range studied. This is typical for IGlu-Na+/K+ of EAAC1 and generally observed under these ionic and pH conditions (
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The voltage dependence of the reverse transport mode of EAAC1, at pH of 7.4 on both sides of the membrane, was weaker than that of the forward transport mode (Fig 4C and Fig D), which is consistent with previous results reported by
Deuterium Kinetic Isotope Effect
Since it is proposed that glutamate transport is associated with the cotransport of a proton rather than a OH- countertransport, we examined the deuterium isotope effect on the steady state and presteady state kinetics of EAAC1. Fig 5 A compares whole-cell current recordings from a voltage-clamped HEKEAAC1 cell exposed to either a D2O-based or a H2O-based bath solution. The steady state glutamate-induced current is reduced by 20% in the presence of D2O compared with H2O, indicating that the steady state turnover of EAAC1 is slightly slowed. To test which step in the transport cycle is affected by the solvent isotope substitution, we performed time-resolved measurements of the transient current component, which are shown in Fig 5 B, for two different voltage-clamped HEKEAAC1 cells. Whereas the time constant of the rising phase is not affected by the substitution of D2O for H2O (0.91 ± 0.07 ms instead of 0.90 ± 0.06 ms with H2O), the time constant of the decaying phase is increased from 10.5 ± 1.2 ms in the presence of H2O to 18.3 ± 2.0 ms in the presence of D2O (Fig 5 C). These results indicate that mainly initial, rapid transporter reaction steps are affected by the solvent isotope substitution.
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To determine if the rate constant and the quantum yield of caged glutamate photolysis are sensitive to substitution of D2O for H2O, we performed the control experiments shown in Fig 3 C. Neither the amplitude nor the decay rate constant of the aci-nitro intermediate absorbance changed when H2O was replaced by D2O, suggesting the absence of a solvent isotope effect on CNB-caged glutamate photolysis.
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DISCUSSION |
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The results presented here provide new insights into the molecular mechanism of proton transport by glutamate transporters. Considering that the formation of a transporter-proton-glutamate complex is necessary for translocation in general, several different kinetic models are possible: (a) the transporter is protonated first, and then glutamate binding takes place (THS); (b) the glutamate anion binds first to the glutamate transporter and the proton binding step follows (TSH); (c) only the protonated form of glutamate is accepted by the transporter, or binding of the proton and glutamate is simultaneous (SHT/SHT-sim); and (d) the binding order of glutamate and the proton is sequential but random (THS/TSH). The models are listed in Table 2 together with the kinetic equations that quantitatively describe KM and Imax as a function of the pH, and they are illustrated in Fig 6 (A and B). For the derivation of these equations, we assumed that, in saturating concentrations of extracellular sodium and intracellular potassium, the turnover number of EAAC1 only depends on the population of the transporter in the state where all of the extracellular ligands are bound (three sodium ions, one proton, and one glutamate anion;
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Using these prerequisites, the predictions of the models are directly comparable with our experimental results. The models TSH and THS/TSH predict that Imax decreases with increasing pH (Fig 6 B), which is not consistent with our data. Therefore, these models can be excluded. For the same reason, we can also exclude that proton binding is rate limiting for the transporter turnover (Table 2, THS slow binding). In contrast, Imax is pH-independent for the other three models. However, models SHT and SHT-sim predict that log(Km) is a linear function of the pH (Fig 6 A). This type of behavior is not found experimentally, allowing us to discard these models. The only mechanism that is in agreement with all the results is THS, assuming that proton binding is fast compared with the transporter turnover. This model is shown in Fig 6 D and correctly predicts that low proton concentrations can be compensated by high glutamate concentrations to fully restore transporter functionality. For this reason, the Imax induced by saturating glutamate concentrations does not depend on the pH. This effect is caused by a stabilization of the protonated form of the transporter upon glutamate binding. Furthermore, the model quantitatively accounts for the weak pH dependence found for the affinity of the transporter for glutamate between pH 6.0 and 8.0 as well as for the drastic increase in KM between pH 8.0 and 10.0. Fitting this model to the extracellular KM values, the pK of the ionizable amino acid residue on the transporter protein can be estimated to be 8.0 in the absence of glutamate (Fig 6A and Fig C). This pK is shifted to more basic values after glutamate binding takes place.
The pK for activation by protons estimated here represents an apparent value that incorporates the following: (a) other rate and equilibrium constants of ion-binding processes on the extracellular side, such as the Na+ binding reaction that we omitted here for the sake of simplicity; and (b) local pH differences related to surface charge effects. The nature of the amino acid residue that binds the proton is unknown. Others (
Based on predictions of the THS model, the decay rate constant of the transient current component, 1/decay (Table 2), is pH-independent at saturating glutamate concentrations (Fig 2 B), whereas 1/
decay decreases at nonsaturating glutamate concentrations, but with a pH-dependent KM (Table 2). All of the predictions drawn from the THS model are in line with our experimentally determined data. Furthermore, at saturating glutamate concentrations, extracellular proton binding does not become rate limiting for the turnover of the transporter or the glutamate translocation step, even at a proton concentration as low as 1 nM. Moreover, the lack of a proton effect on the rate constant of the current rise indicates that protonation of the transporter is not rate limiting for glutamate binding as well. However, this hypothesis requires comparatively high exchange rates of the proton binding residue with protons in the bulk solution or with buffer molecules with a pseudofirst-order rate constant of at least 1,000 s-1. Typical rate constants of proton transfer in aqueous solution are in the range of 1071011 M-1s-1 (
What happens when glutamate is translocated across the membrane and the glutamate and proton binding sites are exposed to the cytoplasm? Both, the data obtained in the forward transport mode and in the reverse mode (KM and Imax) are in the pH range of 6.08.0, which is compatible with a kinetic model that is based on an initial proton binding step of the empty transporter that is followed by glutamate binding, suggesting that the general mechanism of proton transport is similar for forward and reverse transport (Fig 6 D). However, at physiological pH, the apparent affinity for glutamate is reduced 40-fold under reverse transport conditions compared with the forward transport mode, suggesting that the glutamate transporter is asymmetric with respect to its kinetic properties (
The steep rise of the KM value at pH 4.8 in the reverse transport mode of EAAC1 does not fit to the model developed above. At this nonphysiological low pH, the concentration of anionic glutamate is reduced only by 30% with respect to the protonated form. If EAAC1 bound only anionic glutamate, this minor reduction in anionic glutamate would not explain the dramatic decrease in the affinity of EAAC1 for glutamate. 16 µM, pH 4.8, could directly affect the 3-D structure of the transporter, or the functional differences are related to surface charge effects, thus, dramatically changing the kinetic properties of EAAC1. At present, our data do not allow us to differentiate between these possibilities.
In the absence of permeant anions, the Imax value is unaffected by proton concentration. However, if internal SCN- is present, an increase of Imax with decreasing pH was observed. This slight pH dependence of IGlu-anionic may in principle be caused by the following: (a) pH-sensitive rate constants for the transition to the anion-conducting state; (b) a proton dependence of the translocation rate of anions across the membrane; or (c) an additional proton flux through the transporter, such as the one activated by arachidonic acid (rise was never observed in our kinetic experiments (Fig 2). In the third scenario, the voltage dependence of the steady state IGlu-anionic should be affected by the pH because of the additional proton conductance imposed by the pH gradient. This was not observed either, ruling out this possibility as well. Therefore, we favor the second model. The pH dependence of IGlu-anionic is relatively shallow and shows no pronounced inflection point. We speculate that this behavior may be caused by the titration of surface charges of the EAAC1 protein (
The currentvoltage relationship for EAAC1 in the reverse transport mode at physiological pH shows a weaker voltage dependence compared with the forward transport mode, indicating that other steps in the transport cycle with a different voltage dependence become rate limiting under these conditions (Fig 4 D;
The ordered protonglutamate binding model (THS) developed from our experimental data provides further information about the question whether a proton or a OH- is the pH-changing ion that is transported by EAAC1. The existence of a kinetic deuterium effect argues against OH- countertransport. If OH- was countertransported, a kinetic isotope effect of extracellular deuterium should not be observed. Furthermore, the small mass ratio of 0.94 between OH- and OD- is not in agreement with such an effect. In contrast, kinetic isotope effects of 1.53 are found for other proton translocating systems such as bacteriorhodopsin (
How do our results compare to previous studies? It has been suggested that glutamate becomes protonated after it is bound to the transporter. This suggestion was questioned by 107. Thus, the TSH model is not sufficient to explain the proton transport by glutamate transporters.
Apart from L-serine-O-sulfate, cysteine is accepted as a substrate of glutamate transporters (
Physiological Significance
Glutamate is stored in synaptic vesicles at a pH of 55.5 (0.1 U to basic values within a few tens of milliseconds (
1 µM, pH 6.0, (
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Footnotes |
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1 Abbreviation used in this paper: CNB,
-carbonyl-2-nitrobenzyl; EAAC1, excitatory amino acid carrier 1; HEK, human embryonic kidney.
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Acknowledgements |
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We thank Drs. W. Schwarz and F. Weinreich for critical reading of the manuscript, M. Dumbsky for excellent technical assistance, and Dr. H. Wässle for continuous encouragement and support.
This work was supported by the Deutsche Forschungsgemeinschaft (grant No. GR 1393/2-1).
Submitted: 6 July 2000
Revised: 21 August 2000
Accepted: 22 August 2000
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