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J. Biol. Chem., Vol. 278, Issue 4, 2585-2592, January 24, 2003

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Is the Glutamate Residue Glu-373 the Proton Acceptor of the Excitatory Amino Acid Carrier 1?^*

Christof Grewer^§, Natalie Watzke^¶, Thomas Rauen, and Ana Bicho

From the  Max-Planck-Institut für Biophysik, Kennedyallee 70, Frankfurt D-60596 and  Westfälische Wilhelms Universität Münster, Institut für Biochemie Wilhelm-Klemm-Strasse 2, Münster D-48149, Germany

Received for publication, August 5, 2002, and in revised form, September 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glutamate transport by the neuronal excitatory amino acid carrier (EAAC1) is accompanied by the coupled movement of one proton across the membrane. We have demonstrated previously that the cotransported proton binds to the carrier in the absence of glutamate and, thus, modulates the EAAC1 affinity for glutamate. Here, we used site-directed mutagenesis together with a rapid kinetic technique that allows one to generate sub-millisecond glutamate concentration jumps to locate possible binding sites of the glutamate transporter for the cotransported proton. One candidate for this binding site, the highly conserved glutamic acid residue Glu-373 of EAAC1, was mutated to glutamine. Our results demonstrate that the mutant transporter does not catalyze net transport of glutamate, whereas Na⁺/glutamate homoexchange is unimpaired. Furthermore, the voltage dependence of the rates of Na⁺ binding and glutamate translocation are unchanged compared with the wild-type. In contrast to the wild-type, however, homoexchange of the E373Q transporter is completely pH-independent. In line with these findings the transport kinetics of the mutant EAAC1 show no deuterium isotope effect. Thus, we suggest a new transport mechanism, in which Glu-373 forms part of the binding site of EAAC1 for the cotransported proton. In this model, protonation of Glu-373 is required for Na⁺/glutamate translocation, whereas the relocation of the carrier is only possible when Glu-373 is negatively charged. Interestingly, the Glu-373-homologous amino acid residue is glutamine in the related neutral amino acid transporter alanine-serine-cysteine transporter. The function of alanine-serine-cysteine transporter is neither potassium- nor proton-dependent. Consequently, our results emphasize the general importance of glutamate and aspartate residues for proton transport across membranes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The uphill transport of negatively charged amino acid substrates catalyzed by high affinity plasma membrane glutamate transporters is driven by the coupled downhill movement of three sodium ions and one potassium ion across the membrane (1-3). In addition, one proton is cotransported with glutamate into the cell (4, 5). Because of this stoichiometry, glutamate transport is electrogenic with a total of two positive charges being translocated to the intracellular side during each completed transport cycle. Whereas glutamate, sodium ions, and the proton are cotransported together in one branch of the reaction cycle (6), the potassium ion is countertransported in the glutamate-independent relocation step of the carrier (7, 8).

Recently, we have investigated the mechanism of proton transport by EAAC1 (excitatory amino acid carrier 1),¹ a neuronal subtype of the glutamate transporter family (9, 10). We (6) and others (11, 12) have demonstrated previously that protonation of EAAC1 creates a high affinity binding site for the amino acid substrate on the transporter. In addition, protonation is required for substrate translocation across the membrane, whereas the relocation of the glutamate-free transporter form requires dissociation of the proton from EAAC1 (6). However, no information was obtained about the molecular nature of the proton binding site on EAAC1 in this previous study. On the basis of the apparent pK_a of the ionizable residue(s) of 6.5-8, and according to a previous interpretation (13), it was hypothesized that the protonation might involve a histidine residue. However, not only histidine residues, but also acidic amino acid residues within the transmembrane domain, appear to be important for proton translocation in other proton transporters and pumps (14-17).

In high affinity glutamate transporters at least three acidic amino acid residues are located in putative membrane-spanning regions. These amino acid residues, which are highly conserved in the EAAT family (Asp-398, Glu-404, and Asp-470 in GLT1, which correspond to Glu-367, Glu-373, and Asp-443 in EAAC1, Fig. 1), are critical for the functioning of the transporter as demonstrated by Pines et al. (18). Neutralizing the putative negative charge of these amino acid residues of EAATs by site-directed mutagenesis severely impaired glutamate uptake by the mutant transporters (18). The mutant transporters D398N and D470N are non-functional, whereas Glu-404 mutants showed residual activity that was finally attributed to an electroneutral substrate homoexchange reaction suggesting that the potassium-induced relocation of the transporter is impaired (19). Consequently, the acidic amino acid residue in position 404 appears to be important for the relocation step but not for the initial glutamate translocation reaction of GLT1. Therefore, Glu-404 in GLT1 and homologous amino acid residues in other EAATs could be possible candidates for the proton binding site in glutamate transporters.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Sequence alignment of the highly conserved putative transmembrane-spanning region around Asp-367 and Glu-373 of the EAA and ASC transporters belonging to the EAAT family. Acidic amino acids are shown in red, and the arrow indicates the position of the mutation.

In this study, the charge translocation reaction by the mutant E373Q glutamate transporter EAAC1 (corresponding to E404N in GLT1) was investigated in detail and correlated with the proton transport by EAAC1. Furthermore, it was determined whether Glu-373 contributes to the charge neutralization provided by the empty EAAC1 cation binding sites, and whether its charge moves across the electric field during the charge translocating conformational change of the transporter. Based on the facts that glutamate binding to EAAC1_E373Q is completely pH-independent and homoexchange is unimpaired, we propose that Glu-373 is part of the proton-translocating machinery of EAAC1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular Biology and Transient Expression-- Wild-type EAAC1 cloned from rat retina was subcloned into pBK-CMV (Stratagene) as described previously (20, 21) and was used for site-directed mutagenesis according to the QuikChange protocol (Stratagene, La Jolla, CA) as described by the supplier. The primers for mutation experiments were obtained from MWG Biotech (Ebersberg, Germany). The complete coding sequences of mutated EAAC1 and ASCT2 clones were subsequently sequenced. Rat ASCT2 (22, 23) was subcloned into the EcoRI site of the pBK-CMV vector (Stratagene) for mammalian expression.

Wild-type and mutant EAAC1 and ASCT2 constructs were used for transient transfection of sub-confluent human embryonic kidney cell (HEK293, ATCC number CGL 1573) cultures using the calcium phosphate-mediated transfection method as described previously (24). Electrophysiological recordings were performed between days 1 to 3 post-transfection.

Immunofluorescence-- Immunostaining of EAAC1-expressing cells was performed as described (25). In brief, transfected HEK293 cells plated on poly-D-lysine-coated coverslips were fixed in 5% acetic acid in methanol for 4 min at 20 °C. After several washing steps with phosphate-buffered saline (PBS), they were incubated overnight at 4 °C in 0.1% (v/v) Triton X-100 in PBS in the presence of 0.01 mg/ml affinity-purified EAAC1 antibody (Alpha Diagnostics). Following primary antibody incubation, the cells were rinsed and incubated (1 h) with anti-rabbit IgG conjugated to Cy3 (1:500, Dianova, Germany) in PBS containing 0.1% (v/v) Triton X-100. After washing with PBS and water the cells were affixed with coverslips in Mowiol (Hoechst, Germany). The Cy3 immunofluorescence was excited with a mercury lamp, visualized with an inverted microscope (Zeiss) by using a TMR filterset (Omega) and photographed with a digital camera (Sony).

Electrophysiology-- Glutamate-induced EAAC1 currents were recorded with an Adams & List EPC7 amplifier under voltage-clamp conditions in the whole-cell current-recording configuration (26). The typical resistance of the recording electrode was 2-3 M; the series resistance was 5-8 M. Because of the small glutamate-induced membrane conductance changes (typically <5 nS), series resistance (R_S) compensation had no effect on the magnitude of the observed currents. Therefore, R_S was not compensated. Two different pipette solutions were used depending on whether mainly the non-coupled anion current (with thiocyanate) or the coupled transport current (with chloride) was investigated. These solutions contained (in mM): 130 KSCN or KCl, 2 MgCl₂, 10 TEACl, 10 EGTA, and 10 HEPES (pH 7.4/KOH). Thiocyanate was used because it enhances glutamate transporter associated currents and allows the detection of the EAAC1 anion-conducting mode (21, 27). For the electrophysiological investigation of the Na⁺/glutamate homoexchange mode the pipette solution contained (in mM) 130 mM NaCl/NaSCN, 2 MgCl₂, 10 TEACl, 10 EGTA, 10 glutamate, and 10 HEPES (pH 7.4/NaOH). The currents were amplified with an Adams & List EPC-7 amplifier, low pass filtered at 1-10 kHz (Krohn-Hite 3200), and digitized with a digitizer board (Axon, Digidata 1200) at a sampling rate of 10-50 kHz, which was controlled by software (Axon PClamp). All the experiments were performed at room temperature.

Laser-pulse Photolysis and Rapid Solution Exchange-- The rapid solution exchange was performed as described previously (8, 21). Briefly, substrates were applied to the EAAC1-expressing cell by means of a quartz tube (opening diameter, 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 ~5-10 cm/s, resulting in typical rise times of the whole-cell current of 30-50 ms (10-90%). Laser-pulse photolysis experiments were performed according to previous studies (21, 28). CNB-caged glutamate (Molecular Probes (29)), in concentrations of 1 mM or free glutamate were 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, Göttingen, Germany). 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. The laser energy was adjusted with neutral density filters (Andover Corp.). With maximum light intensities of 500-600 mJ/cm² saturating glutamate concentrations could be released, which was tested by comparison of the steady-state current with that generated by rapid perfusion of the same cell with 1 mM glutamate.

Data Evaluation and Terminology-- For simplicity, the following terminology was used: The glutamate-induced coupled transport current was termed I_/K⁺ in the inward transport mode and in the Na⁺/glutamate homoexchange mode. The uncoupled anion current was named I for the glutamate-dependent component.

Non-linear regression fits of experimental data were performed with Origin (Microcal, Northampton, MA) or Clampfit (Axon Instruments, Foster City, CA) by the use of the following equations: The pre-steady-state currents of the anionic current I, in the presence of SCN) were fitted with a sum of two exponential functions and a steady-state current component (I_ss): I = I₁·exp(t/_rise) + I₂·exp(t/_decay) + I_ss. The pre-steady-state transport currents I_/K⁺ and I (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) + I_ss. Under homoexchange conditions I_ss became zero. The observed time constants of _rise of I were in the range of ~1 ms and therefore similar to the time constants of _decay1 of I_/K⁺ and I. For this reason we named these time constants in the following _fast. A similar time dependence with  ~8 ms was found for the time constants _decay of I, and _decay2 of I_/K⁺ and I. These time constants were named _slow. Dose-response data were fitted with the Hill equation: I = I_max([Glu]/([Glu] + K_m))ⁿ, with n being the Hill coefficient. The K_m as a function of pH was calculated with the following equation: K_m = K_S(K_H + [H⁺])/[H⁺], where K_S and K_H are the apparent affinities for the substrate and the proton.

Each experiment was repeated at least five times with at least three different cells. Error bars represent the error of a single measurement (mean ± S.D.), unless stated otherwise. For some kinetic constants a Student's t test analysis was performed to test for significance of the comparison of WT and mutant transporter data.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mutant glutamate transporter EAAC1_E373Q expressed in HEK293 cells was functionally analyzed by whole-cell current recording experiments. Typical current measurements of wild-type EAAC1 (EAAC_WT) and EAAC1_E373Q after application of saturating concentrations of glutamate by using a rapid solution exchange method are shown in Fig. 2. No steady-state transport currents I_/K⁺ were observed for EAAC1_E373Q (Fig. 2A, n = 10 with 4 cells), whereas under the same conditions EAAC_WT exhibited inward currents with an average amplitude of 35 pA (Fig. 2B, n = 20, 6 cells), in line with previous observations (2). These observations are consistent with results obtained for the homologous mutation of GLT1 (E404N) under steady-state conditions (18). Although glutamate-induced steady-state currents are abolished by the mutation, a rapid inwardly directed transient EAAC1_E373Q current is observed at the time of the glutamate application (Fig. 2A) that is absent in non-transfected control cells (Fig. 2C). This result indicates that EAAC1_E373Q is functionally expressed and mediates glutamate-induced charge movements. Expression of EAAC1_E373Q was confirmed by immunocytochemistry as shown in Fig. 2D, demonstrating the membranous localization of the mutant transporter. The time dependence of the rapid charge movements is too fast to be resolved by the rapid solution exchange method with a maximum time resolution of about 50 ms. Therefore, we used laser-pulse photolysis of caged glutamate, allowing us to generate glutamate concentration jumps within less than 100 µs (21) to study the function of EAAC1_E373Q in more detail.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Steady-state currents and EAAC1 expression. Typical whole-cell current recordings of an EAAC1-E373Q (A) and WT transfected HEK cell (B) after application of 500 µM glutamate (rapid solution exchange during the time indicated by the bar, time resolution ~20-30 ms) determined by using a KCl-based pipette solution and a NaCl-based bath solution I/K+, inward transport mode. Panel C shows a control experiment with a non-transfected cell (Vm = 0 mV, pH 7.3). D, fluorescence micrographs of EAAC1WT (left) and EAAC1E373Q-expressing HEK293 cells (right) immunolabeled for EAAC1. The scale bar represents 20 µm.

Pre-steady-state Kinetics-- EAAC_WT exhibits inwardly directed pre-steady-state currents upon applying a rapid concentration jump of extracellular glutamate I, Fig. 3A). These currents are caused by movement of the cotransported Na⁺ ion(s) into the binding site and, most likely, the electrogenic glutamate translocation reaction (8, 30). Similar rapid inwardly directed charge movements are observed for EAAC1_E373Q (Fig. 3B) when glutamate is photolytically released from 1 mM CNB-caged glutamate. The current rises very rapidly with an average time constant of _rise = 0.3 ± 0.1 ms (n = 8; WT, 0.5 ± 0.1 ms). The decay consists of two components. The time constant for the rapidly decaying component is _fast = 1.2 ± 0.3 ms (n = 4; WT, 0.9 ± 0.1 ms). Thus, the kinetics of the current rising phase and the rapid component of the decaying phase are unchanged compared with the EAAC_WT (p > 0.15).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Pre-steady-state currents. Time-resolved measurement of pre-steady-state transport currents I in the absence of permeant anions (NaCl-based pipette solution containing 10 mM glutamate, homoexchange conditions, NaCl-based bath solution) for WT (right panel) and E373Q (left panel) EAAC1. Glutamate (150 ± 20 µM) was released from 1 mM caged glutamate by laser photolysis at t = 0. The transmembrane potential was 0 mV. The solid lines represent best fits with a sum of three exponential functions to the data (see "Experimental Procedures"). The time constants obtained from the fit for the specific experiments shown here were: fast = 0.9 ± 0.1 ms, slow = 11 ± 0.2 ms (WT), and fast = 0.9 ± 0.1 ms, slow = 25 ± 1 ms (E373Q), respectively. The average values for the time constants are shown in the text.

The second component of the current decay, _slow, was slower than that observed in EAAC_WT. This decay process was previously assigned to the electrogenic glutamate translocation reaction across the membrane. Therefore, the existence of the slow current component indicates that glutamate translocation is still functional in the mutated transporter but slowed compared with EAAC1_WT. Because EAAC_E373Q-mediated currents were generally smaller than those observed for the wild-type, the slowly decaying component could not be quantitatively evaluated for the I signal.

Glutamate-induced Anion Currents-- To obtain quantitative information about _slow, we performed experiments in the anion-conducting mode of EAAC1. It was previously shown for EAAC1_WT that (i) the current mediated by glutamate-induced SCN outward movement I is at least five times larger than the electrogenic transport current, I_/K⁺ (31), and (ii) the time constants determined for both current components are linked to each other (8). Pre-steady-state current recordings in the presence of intracellular SCN I evoked by photolysis of 1 mM caged glutamate are shown in Fig. 4A. In the forward transport mode (high [K⁺] internal), the EAAC_E373Q current shows predominantly a transient component. The average peak current is I_ps = 100 pA, whereas the stationary current component, I_ss, is only 10 pA (I_ss/I_ps = 0.04 ± 0.01, n = 3). In contrast, the I_ss/I_ps ratio for EAAC1_WT is significantly larger (0.46, p = 0.004, Fig. 4B) (8, 30). The decay of the transient current could be represented with a monoexponential decaying function, within experimental error. The average time constant for this decay of EAAC_E373Q (_slow = 20 ± 2 ms, n = 6) was significantly slower than the decay of the EAAC1_WT (_slow = 7.4 ± 1 ms, n = 6, p < 0.0001). The I_ss/I_ps ratio recorded in the anion-conducting mode is a measure for the turnover rate of the transporter (I_ss/I_ps = k_b·_slow and k_t = k_b (1  k_b·_slow)) (8, 30). Here, k_b is the rate constant for relocation of the glutamate-free transporter. From the I_ss/I_ps ratio, together with the time constant _slow, a maximum steady-state turnover rate constant (k_t) for EAAC_E373Q of about 1.9 s¹ was calculated. This low value, which is almost 20-fold lower than the one determined for EAAC_WT (35 s¹), explains why no steady-state transport currents can be recorded for EAAC_E373Q.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Whole-cell current recordings I from WT and E373Q-expressing voltage-clamped HEK293 cells. Glutamate was photolytically released from 1 mM CNB-caged glutamate with a 340-nm laser flash (400 mJ/cm2) at t = 0. The concentration of photolytically released glutamate was estimated as 150 ± 20 µM. The solid lines represent the best fits to the data according to a sum of two exponential functions. The transmembrane potential was 0 mV at pH 7.3. A and B, forward transport mode; the intracellular solution contained 140 mM KSCN. The time constants for the rise and the decay of the current obtained from the fit (gray line) were: fast = 1.7 ± 0.1 ms, slow = 8.1 ± 0.1 ms (WT), and fast = 1.8 ± 0.1 ms, slow = 35 ± 5 ms (E373Q), respectively. C and D, homoexchange mode; the intracellular solution contained 140 mM NaSCN and 10 mM glutamate. The time constants for the current decay obtained from the fit were: fast = 0.8 ± 0.1 ms, slow = 10 ± 0.2 ms (WT), and fast = 0.7 ± 0.2 ms, slow = 37 ± 10 ms (E373Q), respectively.

Voltage Dependence of EAAC_E373Q Kinetics-- The voltage dependence of the steady-state and pre-steady-state properties of EAAC_WT and EAAC_E373Q are shown in Fig. 5. The rate constant for the current rise of EAAC_E373Q, 1/_fast, increases slightly with increasing transmembrane potential (Fig. 5A). The slope of the log(1/_fast) versus V_m relationship of (0.5 ± 0.3) V¹ is within the experimental error identical to that found for EAAC_WT (1.2 ± 0.3) V¹. In contrast, the voltage dependence of the rate constant for the decaying phase of the current is reversed and about 4-fold stronger, as shown in Fig. 5A. A similar behavior is well documented for the EAAC_WT (8). The slope of the log(1/_slow) versus V_m relationship is (3.9 ± 0.4) V¹, which agrees well with that of EAAC_WT (4.0 ± 0.3 V¹, Fig. 5A).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Voltage dependence of EAAC1 glutamate transport kinetics for WT (open symbols) and E373Q (solid symbols). A, the relaxation rates 1/fast (triangles) and 1/slow (circles) are plotted as a function of Vm. The solid lines represent the results of a linear regression analysis of the log(1/) versus Vm relationship with slopes of (1.2 ± 0.3)·103/mV (WT, fast) and (3.9 ± 0.4)·103/mV (WT, slow). For the mutant the following values were obtained for the slope from the linear regression analysis: (0.5 ± 0.3)·103/mV (E373Q, fast) and (4.0 ± 0.4)·103/mV (E373Q, slow). B, voltage dependence of the glutamate-induced (500 µM) steady-state current of WT (open circles) and E373Q (solid circles) EAAC1 at pH 7.3. The pipette solution contained 140 mM NaSCN and 10 mM glutamate.

The voltage dependence of steady-state currents is shown in Fig. 5B. Because EAAC_E373Q does not catalyze steady-state transport, these experiments could not be performed for I_/K⁺, but were carried out in the presence of intracellular SCN I under homoexchange conditions. For EAAC_E373Q, the currents start to saturate at potentials below approximately 60 mV (Fig. 5B). As demonstrated in Fig. 5A, wild-type EAAC1 shows the same saturation of currents at V_m < 60 mV under homoexchange conditions. Such a saturating behavior has not been found for previous EAAC_WT experiments using the forward transport mode (8).

EAAC_E373Q Currents Are pH-independent-- The experiments described above demonstrate that the E373Q mutation in EAAC only affects the rate of glutamate translocation but not its voltage dependence, suggesting that translocation can also occur in the absence of an acidic amino acid side chain at position 373. To test this hypothesis, we determined the pH dependence of EAAC_E373Q transport kinetics. Fig. 6A shows the dose-response relationship of EAAC_E373Q currents at different extracellular pH values between 7.3 and 10.0. In this pH range EAAC_WT exhibits a 100-fold change in its apparent glutamate affinity (Fig. 6B) (6). In contrast, the apparent affinity of EAAC_E373Q for glutamate is pH-independent. This effect is not caused by an altered intrinsic glutamate affinity of EAAC_E373Q, because at pH 7.3 the K_m of 10 ± 5 µM is not significantly different from that determined for EAAC_WT (K_m = 13 ± 2 µM, p = 0.58). The 100-fold change in K_m has been determined under conditions of EAAC_WT forward transport, whereas the K_m value for EAAC_E373Q was determined under homoexchange conditions. Therefore, we tested if under homoexchange conditions the K_m of EAAC_WT for glutamate becomes pH-independent. At pH 10.0 the K_m value of EAAC_WT increases to 700 ± 250 µM, about 55 times the K_m measured at pH 7.3, indicating that glutamate transport by wild-type EAAC1 is also pH-dependent under homoexchange conditions.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   pH dependence of EAAC1 glutamate transport kinetics for WT and E373Q EAAC1. A, dose-response relationships of glutamate-induced currents for the E373Q mutant (left) and ASCT2 (right) at values for the extracellular pH of 7.3 (open circles), 8.3 (triangles), and 10.0 (closed circles) at Vm = 0 mV. The intracellular solution contained 140 mM NaSCN and 10 mM glutamate (L-alanine for ASCT2, homoexchange conditions). The solid line represents a fit of Michaelis-Menten kinetics to the data with a Km of 10 µM. B, pH dependence of the Km values for glutamate (EAAC1E373Q, circles) and for alanine (ASCT2, solid triangles; ASCT2Q392E, open triangles). The Km values were obtained under homoexchange conditions (140 mM NaSCN, 10 mM glutamate or alanine in the pipette). The dotted line was calculated according to the model shown in Fig. 8 with KS = 5.5 µM and a pKa of the proton acceptor of EAAC1 of 8.1 (see equation under "Experimental Procedures"). C, relative maximum current (Imax) at pH 10.0 normalized to Imax obtained at pH 7.3 for EAAC1WT (black bars), EAAC1E373Q (hatched bars), and ASCT2 (gray bars). The conditions of the experiments were as described in A.

In addition to the pH effect on the K_m value, we determined the maximum glutamate-induced current (I_max) at saturating glutamate concentrations (1 mM) as a function of extracellular pH. As shown in Fig. 6C, the I_max of EAAC_E373Q decreases by 8% at pH 10.0, compared with pH 7.3. A similar slight decrease (25%) is observed for EAAC_WT and was previously attributed to a pH effect on the anion conductance of EAAC1 but not on the transport rate of glutamate, because the maximum transport currents were unaffected by the external pH (6).

As a further control, the pH effect on the neutral amino acid transporter ASCT2 was investigated. In analogy to EAAC_E373Q studied here, the rat ASCT2 contains a glutamine residue in position 392, which is homologous to the glutamate in position 373 of EAAC1 (22). Application of the neutral amino acid L-alanine to ASCT2-expressing HEK293 cells elicited concentration-dependent uncoupled anion currents with an average amplitude of 250 pA (0 mV, n = 8) in the presence of intracellular thiocyanate, sodium ions, and neutral amino acid substrate (I, in homoexchange mode, original data not shown). These results are in agreement with previously published data on ASC transporters (23, 32). The currents saturated with an apparent K_m of 420 ± 40 µM (Fig. 6A), which is somewhat higher than the value of 18 µM reported for mouse ASCT2, which has been expressed in Xenopus oocytes (33). This difference in the observed K_m may be caused by the different expression systems. After increasing the extracellular pH to 10.0, however, the K_m for L-alanine was virtually unchanged (K_m = 410 ± 60 µM, Fig. 6, A and B). Similar results were found for I_max measured at a saturating concentration of 5 mM L-alanine (Fig. 6C). As found for EAAC1 (WT and E373Q), I_max is only weakly dependent on the external proton concentration. This result is in line with the known pH independence of ASCT between pH 7.3 and 10 of the maximum rate of neutral amino acid transport (23, 33).

Generation of a pH-sensitive ASC Transporter-- To test if the glutamine residue in position 392 of ASCT2 is responsible for the pH independence of alanine transport, we generated the reverse mutation of EAAC1_E373Q in this transporter by replacing Gln-392 with a glutamate residue. The pH dependence of the K_m for alanine of ASCT2_Q392E is shown in Fig. 6C (open triangles). In contrast to ASCT2_WT, the K_m of the mutant transporter strongly increases at pH values > 8. The K_m change is 18-fold from pH 7.3 to 10. This K_m increase is reminiscent of that found for EAAC_WT, although the apparent pK_a value is shifted to a slightly more basic value of 9.0 compared with 8.0 found for EAAC.

Absence of Deuterium Isotope Effect on EAAC_E373Q Kinetics-- To further demonstrate the proton independence of EAAC_E373Q function, we performed pre-steady-state kinetic experiments in the presence D₂O (Fig. 7). Clearly, D₂O substitution has no effect on the amplitude of the glutamate-induced current of EAAC_E373Q (Fig. 7, A and C), as well as on the time constant for the rise of the current (1/_fast, Fig. 7, A and B), consistent with data previously obtained for the EAAC_WT (6). However, a 2-fold decrease of the rate constant for the current decay (1/_slow) was observed after the substitution of H₂O by D₂O for EAAC_WT, but this effect was absent for EAAC_E373Q, as demonstrated in Fig. 7 (A and B). For EAAC_E373Q an average value for _slow of 35 ± 2 ms in D₂O and 31 ± 7 ms in H₂O-based buffer solution was observed (n = 10, 3 cells, p = 0.49). These data demonstrate the absence of a significant kinetic isotope effect on EAAC_E373Q.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Absence of a deuterium isotope effect on the steady-state and pre-steady-state kinetics of E373Q EAAC1. The pipette solution contained 140 mM NaSCN and 10 mM glutamate. The transmembrane voltage was 0 mV. A, laser-pulse photolysis of 1 mM caged glutamate with EAAC1E373Q in a D2O (left panel)- and H2O (right panel)-based extracellular buffer solution under homoexchange conditions. B, relaxation rate constants (1/) for EAAC1WT (white bars, 1/fast; hashed bars, 1/slow) and EAAC1E373Q (black bars, 1/fast; gray bars, 1/slow) in the presence of H2O (left) and D2O (right) (n = 10, 3 cells). The caged glutamate concentration was 1 mM. C, steady-state current of E373Q evoked by 1 mM glutamate in D2O (left)- and H2O (right)-based buffer solution (n = 10, 3 cells).

Inhibition of EAAC_E373Q by Extracellular Potassium Ions-- So far, the data suggest that the residue Glu-373 is involved in proton binding in the glutamate translocation step of EAAC1. To further elucidate the transport mechanism, we asked the question: Does Glu-373, after dissociation of the proton, coordinate a potassium ion? To test this hypothesis, the effect of potassium ion concentration on the transport kinetics of EAAC_E373Q was determined. As shown in Fig. 8A, the addition of 130 mM K⁺ to the extracellular side of the transporter inhibits glutamate-induced inward currents I. This inhibition is dependent on the extracellular Na⁺ concentration (Fig. 8B). No inhibition is observed in the presence of 140 mM extracellular Na⁺, a finding that is compatible with the idea that Na⁺ and K⁺ ions prevent each other from binding to EAAC1 (7). These results demonstrate that EAAC_E373Q is not deficient in potassium ion binding but that only the actual relocation reaction of the empty transporter is impaired. The results are supported by the finding that transient currents are still observed in EAAC_E373Q (Fig. 4A), whereas these currents are absolutely inhibited in the EAAC_WT when intracellular K⁺ ions are absent (data not shown). This result indicates that in the presence of intracellular potassium EAAC_E373Q can still carry out the whole transport cycle but with a substantially reduced transport rate. However, when intracellular K⁺ is missing, the transporter is trapped in a state that is not responsive to glutamate.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Interaction of EAAC1E373Q with potassium ions. A, typical whole-cell current recordings with EAAC1E373Q in the presence of 140 mM extracellular Na+ (lower trace, 0 mM K+), 10 mM Na+ (middle trace, 0 mM K+), and 10 mM Na+ in the presence of 130 mM extracellular K+ (upper trace). The currents were induced by application of 200 µM glutamate during the time indicated by the bar with a rapid solution exchange device. The transmembrane potential was 0 mV, and the intracellular solution contained 140 mM NaSCN and 10 mM glutamate (homoexchange conditions). B, Na+ concentration dependence of whole-cell currents normalized to I (140 mM Na+) in the absence (black bars) and presence (gray bars) of 130 mM extracellular K+ (n = 6, 3 cells). The conditions of the experiments were as described in A. At 10 and 20 mM Na+ the paired t test analysis for the K+ inhibition of wild-type and mutant transporter yielded p values of 0.002 and 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In a previous study, we have demonstrated that the proton that is cotransported with glutamate by EAAC1 associates with a binding site on the glutamate-free form of the transporter (6). However, no information was obtained in that study concerning which amino acid residues in the EAAC1 sequence are involved in the binding of the proton. Here, we addressed this question by using site-directed mutagenesis, focusing on the EAAC1 glutamic acid residue 373, which is highly conserved in the EAAT family, but not in the related H⁺-independent ASC transporters that are selective for neutral amino acids. The central result of this study is that the substitution of Glu-373 of EAAC1 with a non-ionizable glutamine residue leads to a transporter that apparently does not interact with protons. Although slowed by a factor of about 2.5 compared with wild-type EAAC1, glutamate translocation catalyzed by the mutant transporter is otherwise unaffected by the E373Q amino acid substitution. The simplest interpretation of these results is that the binding site for protons, which modulates the affinity of EAAC1_WT for glutamate, is permanently occupied in EAAC_E373Q. Therefore, proton release on either the intra- or the extracellular side of the membrane cannot occur, because the glutamine side chain cannot be deprotonated. Because this proton release is essential for the completion of the transport cycle, steady-state turnover of the transporter is impaired. This interpretation is in good agreement with previous reports, demonstrating that the potassium-induced relocation of the glutamate-free transporter is not functional in GLT1 when the EAAC1-homologous glutamate residue Glu-404 was replaced by amino acids with non-ionizable side chain (18, 19). Our results not only underscore the importance of these studies but provide additional information about the molecular mechanism of glutamate transport by investigating partial reaction steps of the transport cycle using pre-steady-state kinetic methods (21, 34).

A Molecular Model for Proton Cotransport by EAAC1-- In the light of the new results we propose a more detailed model for proton cotransport by glutamate transporters. This model is illustrated in Fig. 9 and is based on a cyclic reaction mechanism that incorporates independent reaction steps for glutamate-induced translocation and K⁺-induced relocation of the transporter (7). To create the model as simple as possible, it is assumed that Glu-373 provides the sole binding site for the proton. However, our data do not exclude the possibility that the proton, once bound to the transporter, is shared by several amino acid residues that provide a proton binding network. Such a model was recently proposed for the proton binding to the lac-permease of Escherichia coli (16).

View larger version (46K): [in this window] [in a new window]   Fig. 9.   Simplified four-state cyclic transport model that accounts for the effects of amino acid substitution in position Glu-373 with glutamine of EAAC1. The model is shown for operation in the forward transport mode. The glutamate 373 must be protonated (neutral) to allow glutamate-induced rearrangement of the cation and substrate binding sites to the intracellular side. The K+-induced relocation of the glutamate-free form of EAAC1 is only possible when Glu-373 is deprotonated. In the E373Q transporter this deprotonation cannot take place. Therefore, the relocation of the transporter is impaired, whereas glutamate translocation is not affected by the mutation. To simplify the model, Na+ binding and dissociation reactions were omitted from the kinetic scheme. The apparent pKa values for the proton binding sites on the extracellular and intracellular face of EAAC1 are stated as 8.0 and 6.5, respectively (6), indicating a highly perturbed pKa for Glu-373 compared with free glutamate in solution. It is, therefore, assumed that Glu-373 is more deeply buried in the protein when it is accessible to the extracellular side (shaded area).

In our model, Glu-373 has to be protonated to permit glutamate binding that is followed by a conformational change of the transporter and the final exposure of the glutamate-proton binding sites to the cytoplasm. To complete the reaction cycle and to relocate the binding sites back to the extracellular side, Glu-373 has to be deprotonated and, therefore, negatively charged. Therefore, the transport direction is based on the glutamate concentration- and K⁺ concentration-dependent modulation of the affinity of EAAC1 for protons. Thus, it is essential that the pK_a of Glu-373 be higher than 7 to ensure that the H⁺ binding site is always occupied under physiological conditions when its -carboxylate is accessible from the external side of the membrane. In fact, the apparent pK_a was previously determined to be about 8.0 for EAAC1 (6). Thus, the pK_a of the -carboxylate of glutamate 373 in EACC1 appears to be highly altered compared with the pK_a of glutamate in aqueous solution (~4.2). Consistent with this observation, highly perturbed pK_a values are assumed to be common in catalytically active amino acid residues (35) and are found in membrane proteins that mediate proton transfer, such as bacteriorhodopsin (36, 37), the bacterial reaction center (38) and the F₁F₀-ATP synthase (14). In analogy to these systems, we observed a conformation-dependent modulation of the pK_a of the proton acceptor of EAAC1. In this case, when the binding site for glutamate is exposed to the cytoplasm, the apparent pK_a is shifted to ~6.5 as shown in Fig. 9 (6). Such a pK_a switch is important for the functioning of the EAAC1, because it promotes association of glutamate on the extracellular side and its dissociation on the intracellular side and therefore establishes favorable conditions for inward transport of glutamate (6).

An alternative model for proton transport by native brain glutamate transporters, proposed by Auger and Attwell (39), incorporates separate transport steps for glutamate and the proton suggesting that protons are countertransported in the potassium-driven relocation step of the glutamate transporter (39). Obviously, such a model would explain the absence of an effect of the E373Q amino acid substitution on the glutamate translocating branch of the half cycle, assuming that Glu-373 is always protonated independent of the state of the transporter. However, this model is not consistent with some of the experimental data. First, the authors assumed that changing the external pH has no effect on the transporter kinetics when homoexchange conditions are applied. This assumption is in clear contrast to the data presented here, showing that the extracellular proton concentration strongly modulates glutamate affinity in both the forward and the homoexchange mode. Second, no effect of proton concentration on the amplitude of synaptically evoked transport and anion currents was found (39), which was taken as further evidence that [H⁺] does not affect glutamate translocation. Considering the model shown in Fig. 9, this result is not surprising because reduced proton concentration can be compensated by increased glutamate concentration. Therefore, at saturating [glutamate], I_max becomes pH-independent (6). The glutamate concentration in the synaptic cleft after release is not precisely known, but probably is high enough to saturate the glutamate binding site on EAATs even at elevated extracellular pH. Third, Watzke et al. (6) demonstrated recently the existence of a deuterium isotope effect on the glutamate translocation rate of EAAC1, which indicates that the glutamate-translocating reaction branch of EAAC1 is, in fact, involved in proton transport. For these reasons, the H⁺ exchange model presented in Ref. 39 should be re-evaluated.

At present, two models of the transmembrane topology of EAATs are discussed in the literature. One model proposed for the EAAT sequence region around Glu-373 a membrane-spanning -helix structure (40), whereas the other model suggests a pore-loop-like structure that dips into the membrane from the extracellular surface (41, 42). In both topology models Glu-373 is located in a fairly hydrophobic, yet somewhat water accessible segment of the transporter that has been also implicated to contribute to the binding of cotransported ions other than the proton. For example, the amino acid residues Asp-367 (highlighted in Fig. 1) and Tyr-372 (Asp-398 and Tyr-403 in GLT1) were proposed to participate in binding of Na⁺ and K⁺, respectively (18, 43). In addition, some cysteines that substitute amino acid residues close to Glu-373 in the EAAT sequence can be protected from sulfhydryl-reactive reagent labeling by EAAT substrates and competitive inhibitors (41). Taken together, these data suggest that the highly conserved stretch of amino acids shown in Fig. 1 may form part of the permeation pathway for the substrate and co- or countertransported ions. In agreement with these structural interpretations, we found a highly perturbed pK_a of Glu-373, indicating that this amino acid residue is physically located in an environment that is much different from a free carboxyl group in aqueous solution. It can be speculated that Glu-373 is partially buried in the low dielectric interior of the transport protein, as illustrated in Fig. 9, but still accessible for proton binding from the extracellular water phase. Consistently, EAAC1 in which glutamate in position 373 is substituted by a cysteine residue is unaffected by extracellularly applied positively and negatively charged sulfhydryl reagents (see Ref. 44)² and becomes only accessible when extracellular Na⁺ is removed (44), supporting the view that in the presence of Na⁺ this position is not easily accessible from the aqueous phase. Furthermore, the neighboring amino acid residue Tyr-372 (Tyr-403 in GLT1) was proposed to be alternately accessible to either side of the membrane, depending on the conformation of the carrier (44), consistent with the alternate accessibility model for Glu-373 proposed here.

Interaction of EAAC1_E373Q with Potassium Ions-- Our model discussed above for the E373Q mutation does not exclude the possibility that Glu-373 binds H⁺ in the glutamate translocation reaction and K⁺ in the relocation reaction of the glutamate-free form of EAAC1. In fact, it was previously reported that the EAAC_E373Q-analogous GLT1_E404D mutant is defective in potassium ion binding and/or potassium ion countertransport (19), supporting the idea of an alternating H⁺/K⁺ binding. This interpretation was based on the observation that GLT1_E404D is not able to catalyze reverse transport induced by application of extracellular K⁺ (19). However, using this experimental approach it is not possible to rule out that K⁺ still binds to the transporter but does not induce the conformational change associated with the relocation reaction. Here, we tested the latter possibility by determining if Na⁺/glutamate homoexchange is inhibited by increasing external [K⁺]. Inhibition by potassium ions was found, indicating that K⁺ can still bind to the mutant transporter. In contrast, K⁺-induced relocation of the EAAC1 binding sites is impaired, which is in line with the observations made by the Kavanaugh and Kanner groups (19). Our results suggest that the acidic amino acid residue Glu-373 is not essential for the binding of K⁺ to EAAC1. The negative charge on Glu-373, however, controls the rate of the K⁺-dependent conformational change of the K⁺ branch of the transport cycle. It can be speculated that Glu-373 is important for counterbalancing the positive charge of the potassium ion to facilitate its movement across the low dielectric membrane barrier. Therefore, Glu-373 may provide one of the two negative charges that are involved in this reaction (21). These negative charges are essential for the functioning of the transporter, because they also electrostatically compensate the three positive charges moved along with glutamate in the substrate translocation reaction.

Recently, a new model was proposed to explain the apparently defective K⁺ interaction of mutant glutamate transporters (45). This model is based on the finding that the arginine residue in position 446 of EAAC1 is involved in binding of the -carboxylate of glutamate (46). In this report, it was suggested that Glu-373 forms an ion pair with Arg-446 in the absence of potassium ions, whereas it complexes K⁺ in its presence. The results presented here support this model, showing that Glu-373 is important for the K⁺-induced relocation reaction of EAAC1. Extending this model to the glutamate translocating half-cycle of EAAC1, one can speculate that Glu-373 forms an ion pair with Arg-446 only in the absence of protons and glutamate. To allow glutamate binding, the ion pair must be destabilized, which is accomplished by binding of H⁺ to Glu-373, thus making Arg-446⁺ available for associating with glutamate. This model would also explain the sequential binding order for H⁺ and glutamate to EAAC1 that was reported earlier (6).

Importance of Acidic Amino Acid Side Chains for Proton-transporting Systems-- The results presented here emphasize the general importance of acidic amino acid residues for proton transport across membranes. There is compelling evidence for the direct involvement of aspartate and glutamate residues in proton transport by bacteriorhodopsin (15), lac permease (16), F₁F₀-ATP synthase (14), the bacterial reaction center (38), the multidrug resistance transporter EmrE (17), and, presumably, the Ca²⁺ ATPase (47). In all of these transmembrane proteins the important acidic amino acids are localized in the hydrophobic environment of the membrane-spanning part of the protein, and they are characterized by strongly perturbed pK_a values. For the lac permease it has been shown that protonation of Glu-325, which is facilitated by extracellular substrate binding, induces its partitioning into the low dielectric membrane phase (16). The protonated complex is therefore able to undergo the conformational change that leads to exposure of the proton and substrate binding sites to the cytoplasm and subsequent intracellular dissociation of H⁺ and the substrate. This mechanism is remarkably similar to the model that we propose here for proton translocation by EAAC1. In contrast to EAAC1 and lac permease, the transport mechanism of the Ca²⁺ ATPase is based on proton countertransport (47, 48). Protonation of the ATPase occurs most likely at the same acidic amino acid residues that participate in complexing the two calcium ions (47). Although different with regard to the directionality of the proton transport, this mechanism is similar to the mechanism proposed here for EAAC1, because release of the proton is controlled by a pK_a switch that is brought about by conformational changes of the protein. The involvement of acidic amino acid side chains as proton acceptors is critical for such mechanisms, because it ensures the neutrality of the protonated translocation complex.

Mechanism of Amino Acid Transport by ASCT-- Our results also have implications for the understanding of the mechanism of neutral amino acid transport by ASCT2. In agreement with previous suggestions we show that amino acid transport by ASCT2 is associated with a much simpler mechanism than glutamate transport by the EAA transporter family (23). As demonstrated here, changes in the extracellular pH between 7.3 and 10.0 affect neither the translocation rate of alanine across the membrane nor the affinity of the transporter for the substrate. These results indicate that, in contrast to EAAC1, no protons are cotranslocated together with the neutral amino acid substrate. It is, therefore, likely that the proton binding site that is present in EAAC1 is permanently protonated in ASCT2, thus preventing directional proton cotransport. This interpretation agrees well with former reports that demonstrated that the transport rate of glutamine by ASCT is not affected by changes in the extracellular proton concentration at pH values greater than 7 (23, 33), whereas ASCT glutamate transport strongly increases with decreasing pH, suggesting that acidic amino acids are translocated in their protonated, neutral form (33). Furthermore, in contrast to glutamate transport by EAAC1, ASCT transport of neutral amino acids is not associated with intracellular acidification (23). Together, these data demonstrate that ASCT is functionally not affected by the extracellular pH. Interestingly, pH sensitivity can be engineered in ASCT2 by generating the reverse mutation at the homologous position, Q392E. We, therefore, suggest that the distinct difference in transport mechanisms of EAATs and ASCTs is, at least in part, mediated by a simple acidic-to-neutral amino acid exchange in position 373.

	ACKNOWLEDGEMENTS

We thank S. Bröer for kindly providing ASCT2 cDNA, E. Bamberg and H.-J. Galla for constant encouragement and support, E. Grabsch and B. Legrum for help in molecular biology, and A. Becker for subcloning of the ASCT2 cDNA.

	FOOTNOTES

^* This work was supported by the Deutsche Forschungsgemeinschaft (Grants GR 1393/2-2 (to C. G.) and RA 753/1-1 (to T. R.)) and the Fundação para a Ciência e a Tecnologia (PRAXIS XXI/BD/18095/98, fellowship (to A. B.)).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: IonGate Biosciences GmbH, Paul-Ehrlich-Strasse 17, Frankfurt/Main D-60596, Germany.

^§ To whom correspondence should be addressed. Tel.: 49-69-6303-336; Fax: 49-69-6303-305; E-mail: grewer@mpibp-frankfurt.mpg.de.

Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M207956200

² C. Grewer, N. Watzke, T. Rauen, and A. Bicho, unpublished results.

	ABBREVIATIONS

The abbreviations used are: EAAC1, excitatory amino acid carrier 1; CMV, cytomegalovirus; PBS, phosphate-buffered saline; WT, wild-type; CNB, -carboxy-O-nitrobenzyl; ASC, alanine-serine-cysteine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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