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J. Biol. Chem., Vol. 281, Issue 15, 10263-10272, April 14, 2006

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Neutralization of the Aspartic Acid Residue Asp-367, but Not Asp-454, Inhibits Binding of Na⁺ to the Glutamate-free Form and Cycling of the Glutamate Transporter EAAC1^*

From the Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33136

Received for publication, October 2, 2005 , and in revised form, January 17, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na⁺ ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, Asp-367 and Asp-454, in Na⁺ cotransport. To test the effect of charge neutralization mutations in these positions on Na⁺ binding to the glutamate-free transporter, we recorded the Na⁺-induced anion leak current to determine the K_m of EAAC1 for Na⁺. For EAAC1_WT, this K_m was determined as 120 mM. When the negative charge of Asp-367 was neutralized by mutagenesis to asparagine, Na⁺ activated the anion leak current with a K_m of about 2 M, indicating dramatically impaired Na⁺ binding to the mutant transporter. In contrast, the Na⁺ affinity of EAAC1_D454N was virtually unchanged compared with the wild type transporter (K_m = 90 mM). The reduced occupancy of the Na⁺ binding site of EAAC1_D367N resulted in a dramatic reduction in glutamate affinity (K_m = 3.6 mM, 140 mM [Na⁺]), which could be partially overcome by increasing extracellular [Na⁺]. In addition to impairing Na⁺ binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which Asp-367, but not Asp-454, is involved in coordinating the bound Na⁺ in the glutamate-free transporter form.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Excitatory amino acid carrier 1 (EAAC1)³ belongs to the excitatory amino acid transporter (EAAT) family, which consists of five members, EAAT1 (GLAST) (1), EAAT2 (Glt-1) (2-4), EAAT3 (EAAC1) (5), EAAT4 (6), and EAAT5 (7). The major function of glutamate transporters in the central nervous system is to remove glutamate from the synaptic cleft to prevent the glutamate concentration from reaching neurotoxic levels (8, 9). Glutamate/aspartate transporters move glutamate from the extracellular space into the cell against its own transmembrane concentration gradient by coupling transport to the downhill movement of Na⁺ and K⁺ ions across the membrane (5). The stoichiometry of this coupling is movement of 1 glutamate^-, 3Na⁺, and 1 H⁺ into the cell and 1 K⁺ out of the cell (9-12). According to this stoichiometry, glutamate transport is electrogenic with a total of two positive charges being translocated to the intracellular side during each transport cycle.

Important questions that are still unresolved regard the translocation pathways and the location of binding sites of the substrate and the co- and countertransported ions. One breakthrough finding toward localizing the glutamate binding site was that an arginine residue (Arg-446) in EAAC1 is responsible for the coordination of the negatively charged -carboxylate group of glutamate (13). Replacement of this amino acid by cysteine changes EAAC1 from an acidic amino acid transporter to a neutral amino acid transporter indicating that Arg-446 is absolutely necessary for the binding of the negatively charged -carboxylate group of glutamate to EAAC1. Although this involvement of Arg-446 in glutamate binding was confirmed in the recently published crystal structure of a bacterial glutamate transporter homologue (14), GltPh, possible binding sites for the co-transported cations were not resolved in this structure. Because the mechanism of glutamate transport by EAAC1 is assumed to be based on charge compensation (15), it is likely that negatively charged residues in the transmembrane segments of the transporter contribute to the binding of these cations (16, 17). Several acidic amino acid residues are highly conserved in the transporter family (16). One of them, Glu-373 in EAAC1, was proposed to be responsible for proton binding (18). Furthermore, it was suggested that this amino acid residue is also involved in the K⁺ interaction with the transporter (19).

In another report, Pines et al. (16) hypothesized that two aspartate residues of Glt-1 corresponding to Asp-367 and Asp-439 in EAAC1 may be involved in the binding and/or translocation of one or more of the cotransported cations. However, these authors did not show clear experimental evidence to support this hypothesis besides the fact that transporters with charge neutralizations in these positions were not functional.

Here, the requirement of aspartate residues Asp-367 and Asp-454 for the transport function of EAAC1 was studied in more detail. Both of these residues are highly conserved among members in the EAAT family (Fig. 1) and are localized in the transmembrane domain of the transporter, according to the GltPh structure (14). Charge neutralization of these two residues resulted in transporters that were still able to bind Na⁺, but did not catalyze glutamate transport. Whereas the D454N mutation had only little effect on the apparent affinity of Na⁺ binding to the empty transporter, EAAC1_D367N showed strongly decreased apparent affinity for this Na⁺ binding step. Mutation of the corresponding amino acid residue Asp-386 to asparagine (Fig. 1) in the related neutral amino acid transporter ASCT2 (alanine-serine-cysteine transporter 2), which has a much higher affinity to Na⁺ than EAAC1, resulted in a mutant transporter with a very similar behavior to that of EAAC1_D367N, namely, the affinity for Na⁺ was dramatically decreased. Based on these results, we propose that Asp-367 in EAAC1, but not Asp-454, is involved in the binding of Na⁺ to the glutamate-free form of the transporter.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Sequence alignment of the highly conserved region around Asp-367 and Asp-454 of EAATs and ASCTs belonging to the SLC1 (solute carrier 1) family. The conserved aspartic acid residues Asp-367 and Asp-454 are shown in red.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Electrophysiology—Substrate-induced transporter currents were recorded with an Adams & List EPC7 amplifier under voltage-clamp conditions in the whole cell current-recording configuration. The typical resistance of the recording electrode was 2-3 M; the series resistance was 5-8 M. Because the currents induced by substrate or cation application were small (typically <500 pA), series resistance (R_S) compensation had a negligible effect on the magnitude of the observed currents (<4% error). Therefore, R_S was not compensated. For the experiments in the forward transport mode, the extracellular solution contained (in mM): 140 NaCl, 2 CaCl₂, 2 MgCl₂, and 30 HEPES, pH 7.3. Na⁺ was replaced by NMG⁺ (N-methylglucamine) in Na⁺-free solutions. Two different pipette solutions were used depending on whether mainly the non-coupled anion current (with thiocyanate) or the coupled transport current (with chloride or gluconate) was investigated (20). These solutions contained (in mM): 130 KSCN or KCl/K-gluconate, 2 MgCl₂, 10 EGTA, and 10 HEPES (pH 7.4, KOH). Thiocyanate was used because it enhances glutamate transporter-associated currents and it allowed us to determine the effect of the Na⁺ concentration on the leak anion conductance mode (20).

For the electrophysiological investigation of the Na⁺/glutamate exchange mode the pipette solution contained (in mM) 140 NaCl/NaSCN, 2 MgCl₂, 10 EGTA, 10 glutamate, and 10 HEPES (pH 7.4, NaOH) (21). It was shown previously that application of extracellular glutamate in the exchange mode results in a redistribution of glutamate binding sites within the membrane associated with permanent activation of an anion current (22, 23). This permanent anion current was used as a tool to study the behavior of mutant transporters in the exchange mode. The currents were low pass filtered at 3 kHz, 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.

Rapid Solution Exchange and Laser-pulse Photolysis—Rapid solution exchange was performed as described previously (15). Briefly, substrates were applied to the EAAC1- or ASCT2-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 20-50 ms (10-90%). Laser-pulse photolysis experiments were performed according to previous studies (15, 22). 4-methoxy-7-nitroindolinyl-caged glutamate (24) (Tocris) in concentrations of 1-4 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 at a distance of 300 µm from the cell. 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 a glutamate concentration that saturated the respective mutant transporter.

Data Analysis—Non-linear regression fits of experimental data were performed with Origin (Microcal Software, Northampton, MA) or Clampfit (pClamp8 software, Axon Instruments, Foster City, CA). Data points are given ± S.D. Pre-steady-state currents were fitted with sums of two exponential terms. Dose-response relationships of currents were fitted with a Michaelis-Menten-like equation, yielding K_m and I_max. The glutamate-free transporter form has a relatively low affinity for extracellular Na⁺. Therefore, it was not possible to saturate the binding site on this transporter form within the [Na⁺] concentration range accessible. For this reason, we estimate the error associated with the determined K_m values to be at least ±20%. This is especially relevant for the data on the D367N mutant transporter, for which it was not possible to reach half-saturating concentrations (the current-[Na⁺] relationship was almost linear). Therefore, only a crude estimate of the K_m of this mutant for Na⁺ can be given.

View larger version (8K): [in this window] [in a new window]   SCHEME 1

(Eq. 1)

(Eq. 2)

Here, K_I and K_Na⁺ are the apparent dissociation constants of the inihibitor and Na⁺, respectively. It is clear that in the presence of saturating concentrations of inhibitor Equations 1 and 2 are identical. Thus, when obtaining the apparent affinity of the transporter for sodium by using the inhibitor-induced current or Na⁺-induced current the same result should be obtained.

View larger version (13K): [in this window] [in a new window]   FIGURE 2. Application of Na+ to EAAC1-expressing cells activates leak anion current. A, typical Na+-induced inward current ([Na+] = 140 mM, application time is indicated by the gray bar) in the presence of 130 mM intracellular SCN-. B, co-application of 20µM TBOA (white bar) with Na+ leads to inhibition of 90% of the Na+-induced current. C, current recording from the same cell, but TBOA was applied in the continuous background of 140 mM Na+. D, typical current recording from a control cell not expressing EAAC1. The conditions of the recording were as in A. The transmembrane potential in A-D was 0 mV. E, current-voltage relationship of Na+-induced currents in EAAC1WT with SCN- present only in the pipette (closed circles), SCN- only present on the extracellular side (triangles). The open circles show data from control cells not expressing EAAC1 in the presence of intracellular SCN-.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As shown in Fig. 2A, application of 140 mM Na⁺ to an EAAC1-transfected cell in the presence of 130 mM intracellular SCN^- resulted in an inward current (-108 ± 35 pA, from 6 cells) caused by SCN^- outflow. When 140 mM Na⁺ was co-applied with 20 µM TBOA, only 13 ± 3% (n = 5) of the current in the absence of TBOA was observed (see Fig. 2B for a representative curve), showing that the majority (almost 90%) of the Na⁺-induced current was specifically carried by EAAC1 and was not because of nonspecific background conductances. Fig. 2C shows a typical result obtained when 20 µM TBOA was applied in the background presence of 140 mM Na⁺. The apparent outward current is caused by inhibition of the Na⁺-dependent anion leak current as demonstrated previously by us and others (15, 29). To further test the specificity of the Na⁺-induced current, we applied 140 mM Na⁺ to non-transfected control cells (Fig. 2D). The average current induced in these cells was -9 ± 1 pA (from 7 cells). To directly test whether the Na⁺-induced current is carried by SCN^-, we determined the voltage dependence of the inward current. As shown in Fig. 2E, the inward current increased with increasingly negative membrane potentials (closed circles) and did not reverse within the voltage range tested. When the SCN^- concentration gradient was reversed, an outward current was observed at each voltage (Fig. 2E, triangles), with a current-voltage relationship that can be described by the Goldman-Hodgkin-Katz equation (30). Together, these results demonstrate that the EAAC1-containing membrane acts as a specific SCN^- electrode in the presence of Na⁺. In non-transfected control cells, only very little current was observed within the voltage range tested (Fig. 2E, open circles).

Na⁺ Affinity of the Glutamate-free Transporter Form—Next, we applied the [Na⁺] jump method to determine the K_m for Na⁺ binding to the glutamate-free form of wild type EAAC1. As shown in Fig. 3A (closed circles), the anion leak current increased with increasing [Na⁺]. The K_m was estimated as 120 ± 20 mM (n = 3) after correction for the nonspecific component of the leak current (open triangles and dashed line, obtained from non-transfected cells). When the K_m was determined by using the inhibitor method, we obtained a value of 80 ± 20 mM (open circles). These results show that application of both methods allow us to determine the apparent affinity of the empty transporter binding site(s) for Na⁺. It should be noted that the data were fitted with a Hill coefficient of 1, assuming that only one Na⁺ ion binds to the empty transporter. Although using a Hill coefficient of 2 resulted in a worse fit, by evaluating the wild type data we cannot exclude the possibility that two of the three co-transported Na⁺ bind to the empty transporter form.

We chose the [Na⁺] jump method to determine the Na⁺ affinities of transporters with charge neutralization mutations to amino acids in possible Na⁺ binding sites in the following experiments. Fig. 3B shows the relationship between the anion leak current and [Na⁺] for EAAC1_D367N ([Na⁺]up to 500mM was used, note the 2.6-fold expanded concentration scale). This relationship was nearly linear within the [Na⁺] range used (5-500 mM, Fig. 3B, straight dotted line). This means that a [Na⁺] as high as 500 mM is still far from saturating the Na⁺ binding site(s) on EAAC1_D367N. The data can also be fitted by the Hill equation (Fig. 3B, solid line), which allowed us to estimate the apparent K_m for Na⁺ binding to the transporter as 1.9 ± 1.5 M (corrected for nonspecific currents, Fig. 3B, triangles). Although this value is more than 10 times higher than that of wild type EAAC1, it represents only a crude estimate because we were unable to use higher [Na⁺] than 500 mM without damaging the cells. For EAAC1_D367N the nonspecific component was more prominent (42% of the specific current at 500 mM Na⁺, Fig. 3, B and F). In any case, the data clearly show that Na⁺ binds with a much lower affinity to EAAC1_D367N than to EAAC1_WT, as demonstrated by the poor fit of the data with the wild-type K_m fixed to 120 mM (Fig. 3B, curved dotted line).

View larger version (32K): [in this window] [in a new window]   FIGURE 3. [Na+] dependence of leak anion currents. Whole cell current recordings were performed in the homoexchange mode with NaSCN-based pipette solution (140 mM) and at 0 mV transmembrane potential. A-E show leak anion currents as a function of [Na+] for EAAC1WT (A), EAAC1D367N (B), EAAC1D454N (C), ASCT2WT (D), and ASCT2D386N (E), respectively (closed circles). The solid lines represent fits to the Hill equation with a Hill coefficient of n = 1. The straight dotted line in B shows the result from a linear regression analysis, the curved dotted line shows the result of a fit to the Hill equation with a Km fixed to 120 mM (wild-type Km). The dashed lines and open triangles in A-C represent the nonspecific component of the anion leak current, which was determined from non-transfected cells. The open circles in A are results from experiments in which the leak current was inhibited by TBOA. F, comparison of [Na+]-dependent leak anion currents between EAAC1D367N, ASCT2D386N, and non-transfected cells at 500 mM NaCl.

The leak anion current of the two mutant transporters extrapolated to saturating concentrations of Na⁺ was larger than that induced in EAAC1_WT (Fig. 3, A-C). This effect was not caused by increased cell surface expression of the mutant transporters. As shown in Fig. 1 of the supplementary information, cell surface expression (assayed by biotinylation of EAAC1 and subsequent Western blotting) was not increased by the D367N and D454N mutations as compared with the wild type transporter. Thus, it is possible that these mutations alter the anion conductance properties of EAAC1. However, this effect was not further analyzed in this work.

ASCT2 is a neutral amino acid transporter (31, 32), which has an amino acid sequence closely related to those of EAATs. ASCT2 has a very high affinity for Na⁺ (1 mM, see Fig. 3D and Ref. 33). The aspartic acid residue Asp-386 in ASCT2 corresponds to Asp-367 in EAAC1 (Fig. 1). To determine whether Asp-386 has the same effect to ASCT2 as Asp-367 to EAAC1, we mutated this amino acid residue to asparagine. Subsequently, the [Na⁺]-dependent leak anion current was determined (Fig. 3E). The K_m for the first Na⁺ binding of ASCT2_D386N was estimated as >200 mM from Fig. 3E. Thus, compared with that of wild-type ASCT2, the affinity for the first Na⁺ of ASCT2_D386N is dramatically decreased. Interestingly, the [Na⁺] dependence of the ASCT2_D386N deviates from Michaelis-Menten-type behavior, especially at low [Na⁺] (<50 mM), where we observed a small, but significant outward current. Because of the small size of this current, this effect was not further studied in this work, but it could point to an additional Na⁺ binding site on the glutamate-free transporter form. We also tested the ASCT2 transporter with the D473N mutation, corresponding to D454N in EAAC1. The apparent dissociation constant of Na⁺ from the empty transporter was 5 ± 2 mM, showing that the effect of this mutation on Na⁺ binding is minor.

Affinities for Amino Acid Substrates and Competitive Inhibitors—The Glt-1 analog of EAAC1_D367N was previously shown to be defective in glutamate uptake (16). Consistent with this result, application of 100 µM glutamate to EAAC1_D367N (140 mM [Na⁺]) did not result in any measurable current, either in the transport mode, or in the anion conducting mode (n = 10). However, after increasing the glutamate concentration to 5 mM, large inwardly directed anion currents were observed (-870 ± 400 pA, n = 5; a typical current trace is shown in Fig. 4A), demonstrating that the mutant transporter can still bind glutamate and activate the substrate-dependent anion current. The K_m values for substrates of EAAC1_D367N and ASCT2_D386N are listed in Table 1. At 140 mM Na⁺, the K_m of EAAC1_D367N for glutamate was determined as 3.6 ± 0.8 mM (Table 1 and Fig. 4C). This value is about 500 times higher than that of EAAC1 wild type (7.2 ± 1.1 µM, Table 1). The K_m for glutamine of ASCT2_D386N was determined as 105 ± 10 µM at 140 mM NaCl (Table 1, Fig. 4G), which is 4 times higher than that of wild type ASCT2 (25 ± 1 µM, Table 1). These results show that the charge-neutralization mutation in position 367 strongly reduces the apparent affinity for the transported substrate. The K_m values for competitive inhibitors show a similar behavior. At 140 mM NaCl, the K_m of EAAC1_D367N for TBOA was 180 ± 100 µM (Table 1), which is about 35 times higher than that of wild type EAAC1. Under the same conditions, K_m of ASCT2_D386N for benzylserine, a competitive inhibitor of ASCT2 (33), was found to be 3.0 ± 1.2 mM (Table 1), 6 times higher than that of wild type ASCT2. These inhibitor data suggest that part of the effect of low occupancy of the Na⁺ binding site must be directly on substrate binding, and not only on conformational changes following binding that increase the affinity of the transporter for the substrate. These conformational changes, such as substrate translocation, are only possible in the substrate-bound transporter, but not in the inhibitor-bound form of the protein. In contrast to the data obtained for EAAC1_D367N, the K_m of EAAC1_D454N for glutamate was virtually unchanged compared with the wild type transporter (Table 1).

View larger version (27K): [in this window] [in a new window]   FIGURE 4. Determination of the apparent Km values of substrates for EAAC1D367N and ASCT2D386N. Apparent affinities for substrates of EAAC1D367N and ASCT2D386N were determined by recording substrate-induced anion currents in the homoexchange mode (with Na+ and substrate present on the both sides of the cell membrane) at different extracellular [Na+]. Typical anion currents induced by glutamate (for EAAC1D367N) and glutamine (for ASCT2D386N) are shown in A and E, respectively, with Vhold = 0 mV. The apparent Km values for glutamate activation of EAAC1D367N at 50 mM NaCl, 140 mM NaCl, and 300 mM NaCl are (in mM): 8.3 ± 0.7 (B), 3.6 ± 0.8 (C), and 1.8 ± 0.1 (D), respectively. The apparent Km values of glutamine activation of ASCT2D386N at 50, 140, and 300 mM NaCl are (in µM): 242 ± 37 (F), 103 ± 9 (G), and 517 ± 99 (H), respectively.

Na⁺ Affinity of the Glutamate-bound Transporter Form—In addition to at least one Na⁺ ion binding to the glutamate-free transporter, at least one more Na⁺ ion associates with the glutamate-bound form of EAAC1 (22). The apparent affinity of the Na⁺ binding site on the glutamate-bound transporter can be determined at conditions of saturating concentration of glutamate. These conditions lead to a shift in the Na⁺-binding equilibrium of the empty transporter to the fully Na⁺-bound form, thus eliminating the effects of this Na⁺ binding step on the [Na⁺] dependence of the current. Maximum currents (I_max) induced by saturating concentrations of glutamate in EAAC1_WT show a strong dependence on extracellular [Na⁺] (Fig. 5B), in agreement with previous reports (5, 22). The K_m for this Na⁺ ion(s) is 22 mM for wild type EAAC1 (Fig. 5B). Because of the extremely low affinity of EAAC1_D367N for Na⁺, and, thus, for glutamate, it was not possible to perform this experiment by directly measuring I_max. Instead, the I-[Na⁺] relationship of the transporter was determined for EAAC1_D367N at a fixed glutamate concentration of 10 mM, which is not saturating at [Na⁺] lower than 300 mM (see Fig. 4, B-D), as shown by the solid squares in Fig. 5B. We then estimated the I_max at each Na⁺ concentration by extrapolating the K_m versus [Na⁺] curve shown in Fig. 5A. As illustrated in Fig. 5B, the I_max shows strong [Na⁺] dependence. The data can be fitted by the Hill equation with a Hill coefficient of n = 1.9. The K_m for these Na⁺ ions was estimated as 43 ± 11 mM. Compared with wild type EAAC1, this K_m is about 2 times higher, suggesting that the main effect of the D367N mutation is to inhibit Na⁺ binding to the glutamate-free form of EAAC1, but not to the glutamate-bound form of the transporter.

Steady-state Properties—As shown in Fig. 6A, no steady-state transport current was induced by application of 5 mM glutamate to EAAC1_D367N (gluconate was the main intracellular anion, n = 10, 5 cells, V_hold = 0 mV). Because gluconate does not permeate the EAAC1 anion conductance (34) and there was no driving force for anions present to cross the membrane (0 mV), only the coupled transport component of the current should be observed under these conditions. In contrast, application of 1 mM glutamate induced a significant steady-state inward transport current of -80 ± 30 pA in EAAC1_WT (Fig. 6B, n = 10, V_hold = 0 mV, KCl pipette solution). Although no steady-state transport current was induced in EAAC1_D367N (Fig. 6C), there was a transient transport current present upon glutamate application (-35 ± 12 pA, n = 7, Fig. 6A, arrow). This indicates that the reason why steady-state transport current could not be detected was not because there was no mutant transporter expressed in the HEK293T cells studied, but because the replacement of Asp-367 by asparagine has an inhibitory effect on the steady-state transport rate of EAAC1. In the presence of Cl^- instead of gluconate, a small steady-state current was observed, indicating a permeability of EAAC1_D367N for Cl^-.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. [Na+] dependence of the Km for glutamate and the glutamate-induced steady-state anion current, Imax. A, the apparent Km values for glutamate activation of the EAAC1D367N (squares, solid line) and EAAC1WT (triangles, dashed line) anion conductances are plotted as a function of [Na+]. B, anion current as a function of [Na+] determined at 10 mM glutamate (EAAC1D367N, squares; EAAC1WT, triangles). The open symbols represent Imax values, which were calculated by extrapolation according to the Km values shown in A. Because the Km values were not determined at 10 and 20 mM Na+, they were extrapolated according to the solid line shown in A. The dashed line represents the Imax-[Na+] relationship determined for EAAC1WT. The currents were normalized to the current obtained at 140 mM [Na+]. Therefore, the fits of the EAAC1D367N data yield Imax values >1, because the maximum [Na+] of 140 mM used here was not saturating. All experiments were performed in the exchange mode and at 0 mV transmembrane potential.

View larger version (40K): [in this window] [in a new window]   FIGURE 6. Glutamate application induced transient, but not steady-state transport current in EAAC1D367N. Typical whole cell current recordings of an EAAC1D367N (A) and EAAC1WT-transfected cell (B) after application of glutamate under forward transport conditions (anions on both sides of the membrane were gluconate (A) and Cl- (B); Vhold = 0 mV). C, comparison of average transport currents between EAAC1D367N (3 cells) and EAAC1WT (7 cells). D, voltage dependence of transport currents induced by 50 mM glutamate. E, voltage dependence of steady-state transport currents induced by 50 mM glutamate in EAAC1D367N (solid squares) and EAAC1WT-transfected cells (open squares). F, transient transport current in response to application of 5 mM glutamate to EAAC1D367N in the exchange mode.

Kinetics and Voltage Dependence of Pre-steady-state Currents— Whereas the glutamate-induced transient transport current in EAAC1_WT decays within 30 ms (22), the transient phase of EAAC1_D367N transport current decayed much more slowly with a time constant in the range of 200 ms (Fig. 6A). This current decay was slow enough that we were able to measure its decay kinetics by using rapid solution exchange (time resolution of 20-50 ms). The low rate of the decay of the transport current suggested that electrogenic processes, which are rapid in EAAC1_WT, are slowed in EAAC1_D367N. The transient transport current was also present when the experiment in Fig. 6A was repeated in the exchange mode (Fig. 6F), clearly demonstrating that this electrogenic process is associated with the glutamate translocation branch of the transporter.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Voltage dependence of pre-steady-state kinetics. A, time dependence of whole cell anion current induced by application of 5 mM glutamate by rapid solution exchange in a HEK293 cell transfected with EAAC1D367N (forward transport mode, KSCN as pipette solution, Vhold = 0 mV). B, pre-steady-state whole cell current induced by glutamate released from 1 mM 4-methoxy-7-nitroindolinyl-glutamate by laser photolysis in a cell expressing EAAC1WT (same conditions as in A). Note the different time scales of A and B. C, voltage dependence of whole cell currents induced by application of 50 mM glutamate to a cell expressing EAAC1D367N by rapid solution exchange. D, voltage dependence of the relaxation rate constant (1/) as a function of transmembrane potential for the decaying phases of EAAC1D367N (circles) and EAAC1WT (triangles). E, pre-steady-state whole cell anion current induced by glutamate released from 4 mM 4-methoxy-7-nitroindolinyl-glutamate by laser photolysis in a cell expressing EAAC1D367N (KSCN as pipette solution, Vhold = 0 mV). The solid line represents a fit of a 3-exponential function to the data. F, voltage dependence of pre-steady-state whole cell currents induced by glutamate released from 4 mM 4-methoxy-7-nitroindolinyl-glutamate by laser photolysis in a cell expressing EAAC1D367N (KSCN as pipette solution). G, voltage dependence of the relaxation rate constants (1/) as a function of transmembrane potential for the rising phases of EAAC1D367N (800 µM [glutamate], open triangles) and EAAC1WT (solid triangles,5 µM [glutamate]; solid squares, 120 µM [glutamate]) anion currents.

To test whether the rates obtained by rapid solution exchange were not limited by the speed of the solution exchange, we performed laser photolysis of caged glutamate to release free glutamate at the cell surface in the microsecond time range, as shown in Fig. 7, E and F. As in the rapid solution exchange experiments, two phases of the current were observed, a rising phase and a decaying phase (Fig. 7E). The time constant of the decaying phase was similar to the one determined by rapid solution exchange, _decay = 180 ± 50 ms (n = 5). In contrast the rising phase was significantly faster (_rise = 2.1 ± 0.5 ms) than in the rapid solution exchange experiment. _rise was about 4-6 times the value observed for EAAC1_WT at saturating concentrations of glutamate (Fig. 7G). At the glutamate concentration used for the concentration jump (about 0.8 mM), the glutamate binding site of EAAC1_D367N is still far from being saturated. We estimated that only 19% of transporters were in the bound state at this concentration. Thus, it is likely that the rising phase of the anion current was slowed because of the unfavorable pre-equilibrium of the glutamate binding step, being shifted mainly to the unbound form. To test this idea, we performed control experiments with the wild type transporter at a glutamate concentration (about 2 µM) that yields a similar distribution of glutamate-bound and glutamate-free states as in the D367N experiment. The time constant of the rising phase was 4.1 ± 0.1 ms, demonstrating that the slow rise of the EAAC1_D367N anion current was mainly caused by the inability to saturate the glutamate binding site. It should be noted that it is technically not possible to photolytically generate glutamate concentrations required to saturate EAAC1_D367N, because the high concentration of caged compound necessary would generate large inner filter effects (the majority of the light is already absorbed at the surface of the optical fiber and does not reach the cell).

Finally, we determined the voltage dependence of the rate constant of the anion current rise (Fig. 7G). For EAAC1_D367N, 1/_rise slightly decreased with increasing membrane potential. The slope of the log(1/_rise) versus voltage relationship was -1.9 x 10^-3/mV. In contrast, log(1/_rise) increased with increasingly positive voltage for EAAC1_WT at both saturating and non-saturating glutamate concentrations (Fig. 7G) with slopes of 1.7 x 10^-3/mV for both glutamate concentrations used.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this report we studied the effects of charge neutralization of two highly conserved acidic amino acid residues in EAAC1, Asp-367 and Asp-454. Both of these amino acid residues are localized in hydrophobic segments of the transporter sequence and, according to the recently published x-ray structure of the bacterial glutamate transporter GltPh (14), reside in a position close to the center of the lipid bilayer. The two central new findings of this study are that replacement of Asp-367 with asparagine leads to: 1) a dramatic increase of the apparent K_m for Na⁺ binding to the empty transporter, and 2) slowed kinetics of glutamate translocation and transport. The D367N mutation has only a minor influence on binding of Na⁺ to the glutamate-bound transporter form (Figs. 4 and 5). In contrast to D367N, the D454N mutation has very little effect on the affinity of the empty transporter for Na⁺, suggesting that Asp-367, but not Asp-454 contributes to the sodium binding site on the unloaded EAAC1 protein. We confirmed our observations by investigating the effect of the analogous charge neutralization mutation in ASCT2, which has a much higher affinity for Na⁺ than EAAC1. After the corresponding amino acid residue Asp-386 in ASCT2 was mutated to asparagine, the apparent K_m for Na⁺ to this transporter was raised from the micromolar range to about 200 mM (Fig. 2). These data strongly indicate that both Asp-367 in EAAC1 and Asp-386 in ASCT2 are involved in binding of Na⁺ to the empty transporter.

Effect of the D367N Mutation on Substrate Binding and Na⁺ Binding to the Glutamate-bound Form Is Indirect—It is known from wild-type EAAC1 that the occupancy of the Na⁺ binding site on the empty transporter strongly influences substrate binding to the transporter (22). The substrate binding site is only formed when this Na⁺ binding site is occupied. In agreement with this idea, the apparent K_m values for substrates/inhibitors of EAAC1_D367N were strongly increased because the Na⁺ binding affinity was decreased. The apparent K_m for glutamate of EAAC1_D367N at physiological conditions (140 mM NaCl, pH 7.3) was increased more than 700 times compared with that of the wild type transporter (Table 1). At the same conditions, the K_m for the inhibitor TBOA of EAAC1_D367N was increased about 25 times compared with that of wild type (Table 1). ASCT2_D386N showed similar behavior with respect to the K_m values for the substrate glutamine and the inhibitor benzylserine (Table 1).

Like in wild type EAAC1, decreasing [Na⁺] to 50 mM led to an increase in the apparent K_m of EAAC1_D367N for glutamate, whereas increasing [Na⁺] to 300 mM led to a decrease in K_m (Table 1). However, even at 300 mM extracellular [Na⁺], the Na⁺ binding site is only occupied at a maximum of 13% of transporters. Therefore, we expect that if it was possible to increase [Na⁺] even further to saturating values the apparent K_m for glutamate of EAAC1_D367N would be close to that of the wild type transporter. Based on these data, it is likely that the effect of the D367N mutation on the glutamate binding site is indirect, which is in agreement with the x-ray structure of GltPh (14), showing that the glutamate binding site is located at a distance of about 11-12 Å from the aspartate side chain in position 367.

EAAC1 binds extracellular Na⁺ ions sequentially, with at least one Na⁺ binding to the glutamate-loaded form of the transporter (22). The physical location of the binding site for the second Na⁺ on EAAC1 is still unknown. The apparent K_m for Na⁺ binding to the glutamate-bound form of EAAC1_D367N is 43 mM, whereas that of the EAAC1 wild type is in the range of 20 mM (Fig. 5). Therefore, the effect of the mutation on binding of this second Na⁺ ion is much less than that on binding of the first Na⁺. These results suggest that this second Na⁺ binding site is not in direct proximity of Asp-367.

The Asp-367 Mutation Eliminates Steady-state Glutamate Transport— Mutation of Asp-367 to asparagine eliminated glutamate transport activity. This finding is in agreement with results from a previous study by the Kanner group showing that any mutation of the analogous Asp-398 in Glt-1 results in a transporter that cannot take up glutamate (16).

The steady-state transport data were confirmed by pre-steady-state kinetic experiments, showing a dramatic reduction in the rates of specific partial reactions of EAAC1_D367N. For example, we identified one partial reaction (associated with the decay of the anion current) with a time constant of 220 ms. However, the process associated with this time constant is not rate-limiting for steady-state transporter cycling, which is limited by another, even slower partial reaction. The ratio of the steady-state and the pre-steady-state components of the anion current of about 0.15 (details of this method are described in Ref. 15) allowed us to estimate the rate constant of this rate-limiting process as 1.6 s^-1. Thus, steady-state transport is slowed by about a factor of 50 compared with EAAC1_WT, accounting fully for our inability to measure glutamate-induced transport current in this mutant carrier. At this point, it is not entirely clear which partial reactions are slowed by the D367N mutation. However, they are unlikely to be associated with glutamate binding and Na⁺ binding. This interpretation is based on the rapid rise of the glutamate-induced anion current (Fig. 7E), occurring with a time constant similar to that of EAAC1_WT. It is well established for EAAC1_WT that binding of glutamate and Na⁺ are necessary to generate this anion current (22). We can speculate that the reactions that are slowed are associated with structural changes of the transport protein, such as glutamate translocation. In the wild type transporter, the decaying phase of the anion current was proposed to be associated with the glutamate translocation step (15, 29). If this is also the case in EAAC1_D367N, glutamate translocation would be dramatically slowed by neutralization of this residue.

View larger version (16K): [in this window] [in a new window]   FIGURE 8. Hypothetical structural model for Na+ and glutamate binding to EAAC1, based on the three-dimensional structure of the glutamate transporter homologue GltPh (14). The blue sphere represents Na+. Asp-367, assumed to be involved in coordination of Na+, and Arg-446, which is involved in glutamate binding, are shown as balls and sticks. Glutamate is shown as space-fill model. The model also shows the charges of the side chains. The outline illustrates schematic access pathways of Na+ and glutamate to their binding sites (not to scale). The molecular structures lining these access pathways are not known. The figure was generated with PyMol.

In the absence of transported substrate and Na⁺, it is likely that a more open conformation is adopted that allows access of Na⁺ to its binding site from the extracellular side of the membrane. Evidence for significantly different conformations of the Na⁺-bound and the Na⁺-free forms comes from a study using site-directed fluorescence labeling, showing that large fluorescence changes of EAAT3 labeled in position 430 are associated with Na⁺ binding, as well as glutamate binding to the empty transporter form (36). Furthermore, cysteine-scanning mutagenesis analysis of amino acid residues in re-entrant loop 2 of Glt-1 also indicated a conformation change after Na⁺ binding to the substrate-free transporter because several positions in this loop could not be modified by hydrophilic methanethiosulfonate reagents when Na⁺ was present, but could be modified in its absence (37). Although this more open conformation may allow easier access of Na⁺ to its buried binding site, it is likely that a hydrophobic barrier needs to be crossed during binding, because the binding process is associated with transmembrane charge movement. Therefore, Na⁺ traverses part of the transmembrane electric field while accessing its binding site (38).

Taking all these observations together with our proposal of Na⁺ binding close to Asp-367, a structural picture emerges of Na⁺ and glutamate interaction with the transporter, as shown in Fig. 8. We assume that Asp-367 is negatively charged in EAAC1. This assumption is reasonable because we were unable to modify transporter activity with dicyclohexylcarbodiimide, either in the absence or presence of Na⁺ (data not shown). Because dicyclohexylcarbodiimide preferentially reacts with protonated carboxylates (39), it is likely that Asp-367 is not protonated. Lack of the negative charge in position 367 results in a severe reduction of the glutamate transport rate, even with a fully occupied Na⁺ and glutamate binding site, indicating that counterbalancing the positive charge of Na⁺ with the negative charge of the carboxylate ligand is important for transport, but not for access of Na⁺ and the substrate to their binding sites.

According to the physical location of the Na⁺ binding site proposed here in the GltPh structure (14), amino acid side chains other than Asp-367 might be also involved in Na⁺ binding. Interestingly, a buried hydrophilic patch of amino acid side chains in the vicinity of Asp-367 also involves two threonine residues in transmembrane 3, histidine 295 in transmembrane 6, and Asn-365 in transmembrane 7. All these amino acid residues are highly conserved in the mammalian members of the glutamate transporter family and such polar and charged side chains are known to participate in Na⁺ coordination in previously characterized Na⁺ binding sites (40).

In contrast to Asp-367, Asp-454 appears to be not involved in binding of Na⁺ to the empty transporter. It remains to be determined if negative charge on Asp-454 is required for transporter function. Preliminary data indicate that EAAC1_D454N is functionally similar to the wild-type transporter.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant R01-NS049335-02 and Deutsche Forschungsgemeinschaft Grants GR 1393/2-2,3 (to C. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains additional data and supplemental Fig. S1.

¹ Supported by American Heart Association Fellowship 0525485B.

² To whom correspondence should be addressed: 1600 NW 10th Ave., Miami, FL 33136. Tel.: 305-243-1021; Fax: 305-243-5931; E-mail: cgrewer{at}med.miami.edu.

³ The abbreviations used are: EAAC1, excitatory amino acid carrier 1; EAAT, excitatory amino acid transporter; TBOA, DL-threo--benzyloxyaspartate.

	ACKNOWLEDGMENTS

 
We thank Drs. T. Rauen and S. Broer for providing the EAAC1 and ASCT2 cDNAs, and Dr. K. Hartung for helpful comments and suggestions regarding this work.

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