Arginine 445 Controls the Coupling between Glutamate and Cations in the Neuronal Transporter EAAC-1*

The substrate-binding sites in membrane transporters are alternately accessible from either side of the membrane, but the molecular basis of how this alternate opening of internal and external gates is achieved is largely unknown. Here we present data indicating that, in the neuronal electrogenic sodium- and potassium-coupled glutamate transporter EAAC-1, the substrate-binding site and one of the gates, or a residue controlling the gating process, are in close physical proximity. Arginine 445, located only two residues away from a residue implicated in glutamate binding (Bendahan, A., Armon, A., Madani, N., Kavanaugh, M. P., and Kanner, B. I. (2000) J. Biol. Chem. 275, 37436–37442), has been mutated to serine (R445S). Upon expression in oocytes, measurements of l-[3H]-glutamate transport under voltage clamp reveal that the charge/flux ratio for l-glutamate at –60 mV is ∼30-fold higher than that of the wild type. Also, with d-aspartate, R445S exhibits an ∼15-fold increase in this ratio. In contrast to the wild type, the reversal potential of the substrate-dependent currents in R445S shifts to more negative potentials when either the external sodium or potassium concentration is decreased. These findings indicate that these two cations are the main current carriers in the R445S mutant. Introduction of a methionine or a glutamine, but not a lysine, at position 445 gives rise to a phenotype similar to R445S. Therefore, it seems that the elimination of a positive charge in the vicinity of the substrate-binding site converts the transporter into a glutamate-gated cation-conducting pathway.

Glutamate is the major excitatory neurotransmitter in the central nervous system and is removed from the synapse and its surroundings by glutamate transporters. Indeed, investigation of glutamate transporter knockout mice indicates that they play a central role in preventing both hyperexcitability and excitotoxicity (1). The five known eukaryotic glutamate transporters, glutamate transporter-1 (2), glutamate/aspartate transporter-1 (3), excitatory amino acid carrier-1 (EAAC-1) 1 (4), and excitatory amino acid transporters 4 and 5 (5,6), have an overall identity of ϳ50%. A detailed experimental determination of the topological organization of the carboxyl terminal half revealed two oppositely oriented reentrant loops and an extracellular-facing hydrophobic linker region interspersed between two transmembrane domains (7)(8)(9), although some disagreement remains on this issue (10,11). The first insights into how the transporter folds upon itself are provided by a recent study showing that the two reentrant loops are in close proximity (12).
Glutamate uptake is an electrogenic process (13,14) in which the transmitter is cotransported with three sodium ions and one proton (15,16), followed by the countertransport of one potassium ion (17)(18)(19). Glutamate transporters mediate a stoichiometric (coupled) current and a non-stoichiometric (uncoupled) current (20). The uncoupled current, activated in the presence of Na ϩ and Glu Ϫ , is carried by chloride ions (5,6,20). Recently, we and others have shown that the coupled and uncoupled pathways exist in a dynamic equilibrium (21)(22)(23) and that, in EAAC1, the uncoupled but not the coupled current is abolished upon replacing extracellular NaCl with LiCl (24).
Structure-function investigations of glutamate transporters have revealed that many important determinants for substrate interaction reside in domains highly conserved within the family. Transmembrane domain 7, positioned between the two oppositely oriented reentrant loops, contains Tyr-403 and Glu-404, which seem to be involved in K ϩ interaction (19,25). Determinants for Na ϩ and Li ϩ interaction have been found both in transmembrane domain 7 (Thr-400) and in the second reentrant loop (Ser-440 and Ser-443) (24,26). A conserved arginine, at position 477 in glutamate transporter-1 (corresponding to Arg-447 in EAAC1) in transmembrane domain 8, has been shown to play a pivotal role in the interaction with the acidic amino acid substrate and the potassium ion (27). Despite these significant insights, little is known about the molecular basis linking substrate interaction with stoichiometric coupling.
Here we report that, when Arg-445 is mutated to non-positive residues, large glutamate-dependent leak currents but greatly diminished L-[ 3 H]-glutamate uptake rates are observed upon expression of these mutants in oocytes. These leak currents are carried by Na ϩ and K ϩ . It seems, therefore, that a positive charge in the vicinity of the substrate-binding pocket controls the ability of the transporter to couple the fluxes of cations and glutamate.

EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis-An existing construct of rabbit EAAC1 residing in pBluescript SK(Ϫ) (27) was modified to have a stop codon immediately after a 10-histidine tail and to remove a 1.6-kbp 3Ј untranslated region. An NsiI site was engineered into the ␥-aminobutyric acid (GABA) transporter 1 (GAT1) tagged with a 10-histidine tail between this site and the stop codon. The stop codon is located just before the poly-linker of pBluescript SK(Ϫ). Both of these constructs were cut with restriction enzymes NsiI and XhoI, and the EAAC1 sequence obtained therefrom was ligated to the 10-histidine pBluescript SK(Ϫ) background, now devoid of GAT1. This construct, termed E10H, was used as a parent for single-stranded U-DNA generation and subsequent annealing of the antisense mutagenic primer, as described previously (24).
Expression and Uptake in Oocytes-Restriction enzymes StuI and NsiI were used to subclone the mutant sequence from E10H into EAAC1 residing in the vector pOG 2 , where it is flanked by a 5Ј untranslated Xenopus ␤-globin sequence and a 3Ј poly-(A) signal (Ref. 27). The mutant sequence is termed WT-EAAC1 in this study. The subcloned sequence was sequenced on both strands between the two restriction sites noted above. cRNA was transcribed by using mMESSAGEmMA-CHINE (Ambion), injected into X. laevis oocytes, and maintained as described previously (24).
Electrophysiology and Current-flux Measurement-Voltage jumping was performed by using a conventional two-electrode voltage clamp, as described previously (24). Agarose-cushioned micropipettes (1%/2 M KCl) were used on some batches of oocytes to prevent KCl leakage (28). All current-voltage relations represent steady-state net currents ((I bufferϩAA ) Ϫ (I buffer )) elicited by the amino acids (AA) L-aspartate, D-aspartate, or L-glutamate and were analyzed with Clampfit version 6.05 (Axon Instruments). Kinetic parameters were determined by nonlinear fitting to the generalized Hill equation using built-in functions of Origin version 6.1 (Microcal), as described previously (24).
The standard buffer was composed of 130 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , 1.0 mM MgCl 2 , 1.0 mM HEPES/Tris, pH 7.4. In chloride substitution experiments, gluconate salts were used, and an agarose (2%/2 M KCl) bridge was used to connect the recording chamber to the Ag/AgCl ground electrode. In sodium substitution experiments, NaCl was replaced by equimolar concentrations of choline chloride. In experiments where the potassium concentration was varied, the sodium concentration was held constant at 20 mM, and 110 mM NaCl was substituted iso-osmotically with mixtures of choline chloride and KCl so that the potassium concentration ranged between 2.5 and 110 mM.
Current-flux experiments were performed by clamping the oocyte at Ϫ60 mV, stopping the gravity flow, and then adding 100 M D-aspartate or L-glutamate containing D-[2,3-3 H]-aspartic acid (0.39 Ci/mmol) or L-[G-3 H]-glutamic acid (0.40 Ci/mmol), respectively, directly into the bath. After perfusion for 200 s, the gravity flow was re-started, and the substrate was washed out. To remove any nonspecific radioactivity transferred by the pipette, the oocyte was washed briefly in four Petri dishes prior to transferal to scintillation vials. In current-flux experiments, the current was low-pass-filtered at 10 kHz and acquired once each msec with Axoscope1 (Axon Instruments). The amount of charge translocated was determined after baseline subtraction as the areaunder-curve with Clampfit 8 (Axon Instruments) and converted to moles using Faraday's constant.

RESULTS
Altered Substrate Interaction in Mutants at Position 445-We reasoned that residues close in the primary sequence to R447 that have been proposed to interact with the ␥-carboxyl group of di-carboxyl substrates in EAAC1 (27) could also be involved in substrate interaction. This possibility was investigated by subjecting residues in the stretch from D444 to N451 to site-directed mutagenesis. We found that, when the arginine at position 445 (R445-EAAC1) was changed, saturating concentrations of the various acidic amino acid substrates elicited currents of different magnitude. Substitutions at other positions did not have this effect (data not shown).
The WT-EAAC1-mediated currents show no difference in size when elicited by 1 mM of either D-aspartate, L-aspartate, or L-glutamate (Fig. 1). This concentration is saturating for all three substrates on WT-EAAC1 (4), and the similarity in the size of the current agrees with that reported for WT-EAAC1 and its human homologue, EAAT3 (4,29). In contrast, the size of R445S-EAAC1-mediated currents varies dramatically with the substrate (Fig. 1). At Ϫ60 mV, the net currents elicited by 1 mM L-aspartate or D-aspartate are 43 Ϯ 1.6% (n ϭ 10) and 12 Ϯ 2% (n ϭ 10), respectively, of that by L-glutamate (n ϭ 10).
To ascertain that 1 mM also is a saturating concentration for R445S-EAAC1, its apparent affinity (K m ) for these substrates was determined by recording the currents at varying substrate concentrations. In agreement with previous studies on the human homologue of WT-EAAC1 (29), WT-EAAC1 currents elicited by D-aspartate and L-glutamate at Ϫ60 mV are fully saturated at 300 M; the K m for D-aspartate is 37 Ϯ 4.2 M (n ϭ 4; nH ϭ 0.9 Ϯ 0.02), and for L-glutamate, it is 29 Ϯ 6.4 M (n ϭ 4; nH ϭ 0.9 Ϯ 0.05; data not shown). In R445S-EAAC1, the L-glutamate current at Ϫ60 mV is also fully saturated at 300 M, but, in contrast to WT-EAAC1, the K m for L-glutamate is 4.8 Ϯ 1.3 M (n ϭ 4; nH ϭ 0.95 Ϯ 0.05; data not shown). The net currents elicited by D-aspartate in R445S saturate at 10 M, and the concentration eliciting half-maximum current, at Ϫ60 mV, is 0.28 Ϯ 0.02 M (n ϭ 5; nH ϭ 1.0 Ϯ 0.1, data not shown).
Effect of Removing Extracellular Chloride-It is now well established that glutamate transporters mediate an inwardly rectifying current reflecting the stoichiometric forward uptake, as well as a non-rectifying bi-directional uncoupled anion conductance (20,23). By removing chloride from the external recording solution, the outward current resulting from inward flux of chloride should be abolished. In agreement with previous studies (20), the outward current is indeed abolished in WT-EAAC1 when external chloride is replaced by gluconate FIG. 1. Acidic amino acid currents in WT-EAAC1 and the R445S-EAAC1 mutant. Xenopus laevis oocytes expressing either WT-EAAC1 n ϭ 3) or its mutant R445S (R445S-EAAC1) (n ϭ 10) were voltage-clamped and gravity-perfused with normal buffer (see "Materials and Methods") containing no or 1 mM of amino acids D-aspartate (D-Asp), L-aspartate (L-Asp), or L-glutamate (L-Glu). The voltage was stepped from Ϫ25 mV to voltages between Ϫ100 and ϩ40 mV, in increments of 10 mV. Each potential was held clamped for 250 ms, and the steady-state current from 210 to 240 ms at each potential was averaged. The current in the absence of amino acid was subtracted from that in its presence, normalized to the L-glutamate-elicited current at Ϫ100 mV (I), and plotted against the holding potential (V hold ). Values are mean Ϯ S.E. (Fig. 2). Surprisingly, the outward current mediated by R445S-EAAC1 is still observed in the nominal absence of extracellular chloride (Fig. 2), indicating that the outward current mediated by R445S-EAAC1 is carried by ions other than chloride. Experiments in which chloride is partially substituted by the ϳ70fold more permeant thiocyanate anion (24,30) results in an increased outward current (data not shown), indicating that R445S-EAAC1 is capable of entering the uncoupled anion pathway even though the minor change in the reversal potential upon chloride substitution (Fig. 2) indicates that this anion contributes little to the total current.
Effect of Other Residues at Position 445-Additional mutants have been made and analyzed to determine whether it is the removal of the arginine or the introduction of a serine at position 445 which is responsible for both the altered substrate specificity as well as for the chloride-independent outward currents. Substituting Arg-445 with the polar but non-charged glutamine (Fig. 3) or with the bulky and hydrophobic methionine (data not shown) results in currents whose size varies with the substrate, as found for R445S-EAAC1 (Fig. 1). Strikingly, currents are similar in size in a mutant where the arginine is substituted by the positively charged lysine (Fig. 3), just as is the case for WT-EAAC1 (Fig. 1).
The results on the substitution mutants at position 445 raise the possibility that the ability of mutant transporters to exhibit currents which vary with substrate is related to their ability to carry out substrate-elicited outward currents in the nominal absence of chloride. Indeed significant outward currents mediated by both R445Q-EAAC1 (Fig. 4) and R445M-EAAC1 (data not shown) persist in the absence of external chloride, as is the case for R445S-EAAC1 (Fig. 2). In contrast, the outward current mediated by R445K-EAAC1 is abolished when external chloride is replaced by the impermeable gluconate anion (Fig.  4), exactly as observed with WT-EAAC1 (Fig. 2).
Effect of Changing the Extracellular Na ϩ and K ϩ Concentrations-We addressed the question of which ions are carrying the substrate-elicited outward currents in R445S-EAAC1. The observed reversal potential of Ϫ3.8 Ϯ 0.9 mV (n ϭ 17) (Fig. 2) is in between the equilibrium potentials for sodium and potassium, suggesting the possibility that R445S-EAAC1 is permeant to these two cations.
To test this idea, L-glutamate currents mediated by R445S-EAAC1 and WT-EAAC1 were recorded at various extracellular Na ϩ concentrations (Fig. 5, A and B). In the absence of sodium (choline substitution), no glutamate-induced currents were ob-served in either case (Fig. 5, A and B). Significant substrateelicited outward currents were observed above ϳ30 mM Na ϩ in WT-EAAC1, and the reversal potential shifted toward more positive values with decreasing Na ϩ concentrations (Fig. 5B). In contrast, when the extracellular Na ϩ concentration was decreased from 130 to 10 mM, the reversal potential of the R445S-EAAC1-mediated currents shifted from 1.6 Ϯ 1.6 mV to Ϫ39 Ϯ 1.6 mV (n ϭ 3) (Fig. 5A). In addition, both the size and conductance of the substrate gated currents in R445S-EAAC1 increased as the extracellular Na ϩ concentration increased (Fig. 5A), indicating an increased inward flux of sodium as the inward Na ϩ gradient increased. The observed shift in reversal potential is less than the 64.8 mV predicted by the Nernst equation for a selective Na ϩ flux. A possible explanation is that glutamate causes R445S to become more permeant not only to sodium but also to potassium. At 2.5 mM K ϩ and 20 mM Na ϩ , bi-directional substrateelicited currents were observed in R445S that reversed at Ϫ36 Ϯ 1.7 mV (n ϭ 3) (Fig. 5C). If glutamate increases the permeability for potassium, one would predict a shift in this reversal potential toward more positive potentials as the potassium concentration increased. The net currents depicted in Fig. 5C show that this is exactly what happened in R445S. In contrast, the WT-EAAC1-mediated currents remained inward and did not reverse when the potassium concentration increased to 40 or 110 mM (n ϭ 3) (Fig. 5D). This behavior of WT-EAAC1 will be addressed in "Discussion." In R445S, glutamate also elicited currents in lithium medium but not in Tris Plus medium (data not shown) or choline chloride (Fig. 5A). Increasing the MgCl 2 concentration from 1 to 20 mM did not change the substrate-gated currents (data not shown). Changing the pH from 7.5 to 6.5 or 8.5 also did not influence the size and reversal potential of the currents mediated by R445S-EAAC1 (n ϭ 3; data not shown).
Determination of the Charge to Flux Ratio in WT-EAAC1 and R445S-EAAC1-Whereas the currents mediated by R445S-EAAC1 were of equal size or larger than those mediated by WT-EAAC1, the velocity of radioactive uptake by R445S was much lower. The kinetic parameters of uptake by R445S-EAAC1 are: K m ϭ 6.4 Ϯ 1 M and V max ϭ 16 Ϯ 1 pmol/20 min for L-glutamate; and K m ϭ 4 Ϯ 0.5 M and V max ϭ 33 Ϯ 0.9 pmol/20 min for D-aspartate (nH ϭ 1.1 Ϯ 0.2 in all cases; n ϭ 3-5; data not shown). In comparison, the corresponding values for WT-EAAC1 are: K m ϭ 54 Ϯ 7 M and V max ϭ 558 Ϯ 34 pmol/20 min (nH ϭ 1.0 Ϯ 0.05) for L-glutamate and K m ϭ 30 Ϯ
To substantiate the impression that R445S-EAAC1 translocates many more charges than WT-EAAC1 with each molecule of L-glutamate, currents have been recorded simultaneously during radiolabeled uptake on the same oocytes. A holding potential of Ϫ60 mV was chosen to be able to measure significant D-aspartate-elicited currents. Fig. 6A shows a typical result from oocytes clamped at Ϫ60 mV and expressing either WT-EAAC1, R445S-EAAC1, or GAT1 (31). In WT-EAAC1, an uptake of 22.4 pmol D-[ 3 H]-aspartate was associated with a steady-state current of around Ϫ45 nA. Interestingly, the R445S-EAAC1 mutant only takes up 3.4 pmol D-[ 3 H]-aspartate. Despite this diminished uptake, the translocated substrate elicits a current larger than twice that of WT-EAAC1. As a control, we used oocytes expressing GAT1 which do not transport glutamate or aspartate; no substrate-gated currents are measurable in these oocytes, and the D-[ 3 H]-aspartate uptake is 0.62 pmol, similar to uptake by uninjected oocytes (data not shown) which probably reflects nonspecific binding.

DISCUSSION
The recent crystal structures of the lactose permease and the glycerol-3-phosphate transporter, both in their inward-facing conformation, identify the substrate-binding site in an inwardfacing ⌳-shaped structure. According to an alternating access mechanism, this structure is proposed to change into an outward facing V-shaped structure, such that the binding site will be outward facing (32,33). In the discussion that follows, the conformational movements between the outward-and inwardfacing forms that take place during the substrate translocation are referred to as opening and closing of outer and inner gates. The model depicted in Fig. 7 represents the alternating access translocation cycle of the wild-type glutamate transporter EAAC1 (17)(18)(19) and serves as a framework to explain the results from this study. Also indicated is the state at which the R445S mutant is suggested to differ. Translocation occurs when the transporter is loaded with either the sodium/glutamate/proton or with potassium. One full transport cycle represents the sequential translocation of sodium/glutamate/proton in one direction and of potassium in the opposite direction (15, 17). State I represents the empty and outward-facing transporter with a closed inner gate, to which either sodium/glutamate/proton (indicated by Glu for short) or potassium can bind in a mutually exclusive fashion. Under normal physiological conditions, binding of sodium/glutamate/proton (Step 1) brings the transporter into State II, where the potassium-binding site is altered such that potassium no longer can bind. The normal forward uptake cycle for wild type proceeds clockwise from State II, yielding the inward-facing sodium/glutamate/protonbound conformation of State III. Their release to the inside, indicated by Step 3, yields the empty inward-facing conformation of State IV, where the outer gate is closed but where the inner gate now is open. In this empty, inward-facing conformation, potassium or sodium/glutamate/proton can (re-)bind in a mutually exclusive fashion. Under physiological conditions, with low intracellular sodium and higher intracellular than extracellular potassium, it is this latter cation that binds (Step 4) and brings the transporter into State V. The potassiumbound transporter "reorients" to the outside (Step 5), after which potassium is released (Step 6), bringing the transporter back to State I from where a new cycle can start.
Compared with the wild type, the R445S mutant exhibits enhanced glutamate-and aspartate-induced currents despite a greatly reduced radioactive uptake (Fig. 6). Also in contrast to the wild type, the R445S-mediated currents are not carried by chloride (Fig. 2) but rather are carried by sodium and potassium (Fig. 5). These data can be explained in terms of a phenotype in which State III has a defect in the closing rate of the outer gate. Such a defect will result in a situation where both gates are simultaneously open, thereby creating the conducting pathway through which sodium can enter the cell at negative potentials and potassium can exit at positive potentials (indicated by State III for R445S in Fig. 7).
From the conducting glutamate-bound State III, R445S enters a non-conducting state; glutamate may be released either to the inside (Step 3) or back to the outside by progressing counter-clockwise from State III to State I. Alternatively, the conducting state of R445S may also occur if glutamate binding (Step 1, clockwise) causes both gates to be open simultaneously by premature inner gate opening. The R445S phenotype is described equally well by either possibility. However, we favor the possibility in which the outer gate closure is affected, because if transmembrane domain 8 has a linear sequence in space (9,34), position 445 will be more extracellular than position 447.
The reversal potential of the R445S-mediated currents shifts toward negative potentials when the external sodium or potassium concentration is decreased (Fig. 5, A and C). The shift in reversal potential with potassium for the stoichiometrically coupled process is in the opposite direction (15). The outward currents mediated by wild type are caused by the sodium-and glutamate-gated chloride influx (Refs. 20, 23, 24, 30, and Fig. 2) which is activated during Steps 1 and 2 (35,36). The observed reversal potential shift in wild type with sodium is in the opposite direction to that in the mutant (Fig. 5, A and B). The sodium-dependent glutamate-induced currents in the wild type reflect the sum of the non-reversing coupled currents and the anion currents which reverse at around Ϫ30 mV (20). A reduction of the external sodium concentration is expected to de-crease the size of the coupled current as well as the contribution of the reversing anion current, thereby shifting the reversal potential of the summed current to more positive values. The shift of the reversal potential of the mutant with sodium is probably not solely determined by the altered driving force, but is also likely to be influenced to some extent by the fact that sodium itself enables glutamate to induce the currents.
When the external potassium concentration is increased, the otherwise bi-directional currents mediated by wild type are now exclusively inward at all potentials (Fig. 5D). This can also be easily explained in the framework of the model presented in Fig. 7. At negative potentials, the glutamate-elicited inward currents mediated by wild type result from its clockwise progression through the cycle. Increasing the external potassium concentration in the presence of glutamate (which are the conditions of the experiment in Fig. 5D) will increase the probability that potassium rather than glutamate will bind to State I. Because their binding is mutually exclusive, the transporter counter will be brought clockwise into State VI (Fig. 7), and the glutamate-elicited inward current will be diminished. At positive potentials, the elevated potassium concentration gives rise to outward current because of reverse transport (15,17,37) taking place during the counter-clockwise progression from Step 6 to Step 1 (Fig. 7). These outward currents are decreased when glutamate is added to the outside, again because of the mutually exclusive binding of potassium and glutamate. Subtracting the currents in the absence of external glutamate (smaller inward current and larger outward current) from those in its presence (larger inward current and smaller outward current) yields the inward currents shown for wild type (Fig. 5D).
Two types of uncoupling have been inferred in ion-coupled transporters. In one type there is a defect in the sequential binding of the driving ion and the driven substrate, whereas in FIG. 7. The transport cycle of wild type and R445S. The empty transporter (I) is illustrated with a binding site for the one potassium and another for the three sodium ions/one glutamate/one proton (illustrated by Glu), according to the established stoichiometry. The uncoupled anion current is not indicated. The observed uncoupled sodium and potassium current mediated by R445S is indicated by the trailing directional arrows in III which, in R445S, reflect impaired outer gate closure. States are indicated by Roman numerals, whereas the steps connecting these states are indicated by Arabic numerals. The detailed description is given in "Discussion." the other, the binding sites are simultaneously accessible from either side of the membrane. An example of the first case is provided by the bacterial multidrug/proton exchanger EmrE, in which glutamate-14 is involved in the sequential binding of the proton and the positively charged drug (38). Mutating glutamate-14 to aspartate decreases the apparent proton affinity and results in a decreased ability of the proton to release the drug (38). The second case has been suggested to occur in mutants of the proton-coupled lactose permease that exhibit a constitutive proton-leak, which can be blocked by substrate (39) or in mutants in which the substrate elicits a protonleakage (40). This latter scenario is phenotypically similar to the R445S mutant. Importantly however, a significant novel feature of our study is that the determinant, which causes glutamate to open both gates simultaneously, may be close to its binding site. Arginine 447, close to the arginine 445 investigated here, has been inferred as a binding residue of the glutamate ␥-carboxyl group (27). Moreover, recent studies show that, when the cysteine occupying the equivalent position of arginine 447 in the neutral amino acid transporter 2, is replaced by arginine, the transporter is converted into an acidic amino acid transporter. 2 In addition, in the neutral amino acid transporter 1, threonine occupies the equivalent position and its substrate specificity can be converted into that of the neutral amino acid transporter 2 by replacing it with cysteine, and vice versa. 2 Thus, even though a crystal structure of a glutamate transporter is not yet available, there is strong evidence that position 447 of EAAC1 is indeed part of the substratebinding site. Arginine 445 is uniquely positioned (only two residues away) to modulate glutamate-dependent gating. Thus, the substrate-binding site and one of the gates, or a residue controlling the gating process, are in close physical proximity.
The V max and K m values of transport by the R445S mutant are greatly reduced compared with wild type. As the K m is the composite of the several rate constants associated with various steps, one possibility is that a rate-constant associated with a step subsequent to binding is lowered in this mutant. Indeed, it could be decreased to such an extent that the affected step becomes rate-limiting for the transport cycle, thus causing the observed reduction in V max .
In contrast to wild type, the maximum substrate-elicited currents (I max ) mediated by the R445S mutant vary with the substrate (Fig. 1), and the major part of this current is uncoupled (Figs. 5 and 6). One attractive possibility is that L-glutamate, L-aspartate, and D-aspartate differ in their residency times at State III (Fig. 7). As long as the substrate resides in its binding site, the uncoupled flux continues, and its magnitude will vary according to how long it remains bound. Another possibility is that the substrate-binding pocket is physically linked to the gating mechanism, and the different dimensions of the substrates influence the rate by which the outer gate can close.
The net outward substrate-dependent currents mediated by other mutants where arginine 445 has been changed to an uncharged residue are also not carried by chloride (Figs. 2 and  4). Therefore, it appears that, in the absence of a positive charge at position 445, gating is impaired concomitant with differences in the maximal substrate-elicited currents (Figs. 1  and 3). It is not clear, however, why a positive charge at position 445, which is highly conserved in the glutamate trans-porter family is essential for optimal gating. One possibility is that glutamate binding causes the arginine at position 445 to move, thereby enabling it to interact physically with a negatively charged amino acid residue. The formation of such a salt bridge could induce closure of the outer gate directly, but it is also possible that this occurs by means of an indirect conformational change. Site-directed mutagenesis of conserved negatively charged residues, in conjunction with the type of experimental approach used here, may shed further light on the molecular mechanism of ion-coupled transporters.