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J. Biol. Chem., Vol. 277, Issue 16, 13494-13500, April 19, 2002

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Distinct Conformational States Mediate the Transport and Anion Channel Properties of the Glutamate Transporter EAAT-1^*

Renae M. Ryan and Robert J. Vandenberg^§

From the Department of Pharmacology, University of Sydney, Sydney, New South Wales 2006, Australia

Received for publication, October 16, 2001, and in revised form, January 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na⁺, 1 H⁺, and the counter-transport of 1 K⁺ ion. In addition to coupled ion fluxes, glutamate and Na⁺ binding to the transporter activates a thermodynamically uncoupled anion conductance through the transporter. In this study, we have distinguished between these two conductance states of the EAAT-1 transporter using a [2-(trimethylammonium)ethyl]methanethiosulfonate-modified V452C mutant transporter. Glutamate binds to the modified mutant transporter and activates the uncoupled anion conductance but is not transported. The selective alteration of the transport function without altering the anion channel function of the V452C mutant transporter suggests that the two functions are generated by distinct conformational states of the transporter.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system, and the role of the excitatory amino acid transporters (EAATs)¹ is to regulate synaptic glutamate concentrations to maintain dynamic signaling between the presynaptic and postsynaptic membranes and also to prevent a build-up of toxic levels of glutamate. The energy used to drive the uphill transport of glutamate into the cell against a significant concentration gradient is derived from the coupling of transport to the co-transport of 3 Na⁺ and 1 H⁺ and the counter-transport of 1 K⁺ for each glutamate molecule (1, 2). In addition to these coupled ion fluxes, glutamate bound to the transporter activates a thermodynamically uncoupled anion conductance through the transporter (3-5).

Five different glutamate transporters have been identified, and their amino acid sequences were determined from the cDNA sequences. The human glutamate transporters are termed EAAT-1 to EAAT-5 (3, 6, 7), whereas the rat homologs of EAAT-1 and EAAT-2 are termed glutamate/aspartate transporter-1 (8) and glutamate transporter-1 (9) and the rabbit homolog of EAAT-3 is termed excitatory amino-acid carrier-1 (10). The five different transporter subtypes show a high degree of similarity and are likely to form similar structures in the membrane. A number of models for the membrane topology of glutamate transporters have been proposed. Initial predictions of the topology from hydrophobicity analysis of the amino acid sequence lead to three different models with 8, 10, and 12 transmembrane domains (8-10), but more direct structural information has been obtained from method studies of substituted cysteine accessibility (11, 12). The models proposed from these studies both predict six -helical transmembrane domains in the amino-terminal portion of the protein followed by a series of reentrant loops and membrane-associated structures followed by a final -helical transmembrane domain and an intracellular carboxyl-terminal domain.

The reentrant loop and membrane-associated structures are highly conserved in their amino acid sequences, and from mutagenesis studies, it has been proposed that this region forms the pore structure through which glutamate and the various co-transported and counter-transported ions pass. However, it remains to be demonstrated whether any of these residues also interact with anions, which raises the question as to whether the same region of the transporter that mediates the transport process is also responsible for allowing the uncoupled anion conductance. In a recent study (13), we demonstrated that Zn²⁺ ions selectively inhibit the anion conductance with no significant effect on the transport characteristics of the glutamate transporter EAAT-4, which suggests that the transport and anion conductances are mediated by distinct conformational states of the transporter. The Zn²⁺ binding site was found to be in the large second extracellular loop, which is in a region that is distinct from the reentrant loops and transmembrane domains that interact with glutamate and the co-transported and counter-transported cations.

The aim of this study is to further investigate the conformational states responsible for the transport and anion channel functions of the transporter. We have attempted to identify amino acid residues, which when changed to cysteine and modified with the methanethiosulfonate (MTS) class of compounds (14, 15), differentially alter the transport and anion channel properties of the transporter. To achieve this result, amino acid residues, which are accessible to MTS reagents and in close proximity to the pore region but do not directly interact with either glutamate or the co-transported and counter-transported cations, were targeted. Seal et al., (12) identified a number of amino acid residues in a membrane-associated linker region that are accessible to MTS reagents, which appear to satisfy these criteria. In this study, we report [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) modification of the EAAT-1 mutant V452C blocks glutamate transport but does not interfere with glutamate binding to the transporter and activation of the uncoupled anion conductance. The selective disruption of transport while maintaining the anion channel function of EAAT-1 suggests that different conformational states of the transporter mediate the two functions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals-- DL-Threo--benzyloxyaspartate (TBOA) was obtained from Tocris (Bristol, UK), and MTS derivatives were obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). All other chemicals were obtained from Sigma unless otherwise stated.

Site-directed Mutagenesis-- EAAT-1 subcloned into the plasmid oocyte transcription vector (pOTV) was kindly supplied by Susan Amara (The Vollum Institute, Portland, OR). A cysteine-less EAAT-1 (CLE1) was engineered by making conservative substitutions of the three native cysteine residues in EAAT-1 (C186A, C252A, and C375A) using the QuikChange^TM site-directed mutagenesis kit and recommended protocol (Stratagene). Valine 452 was also mutated to a cysteine residue, V452C, using the same protocol. Both CLE1 and V452C were sequenced on both strands by dye terminator cycle sequencing (ABI PRISM, PerkinElmer Life Sciences) at the Sydney University Prince Alfred Macromolecular Analysis Center.

Transporter Expression in Xenopus laevis Oocytes and Electrophysiological Recordings-- The CLE1 and V452C transporter cDNAs were linearized with BamHI and cRNA transcribed with T7 RNA polymerase and capped with 5'-7-methylguanosine using the mMessage mMachine kit (Ambion, Inc., TX). Oocytes were harvested from X. laevis as described previously (16), and 50 nl of cRNA was injected into defoliculated stage V oocytes and incubated in standard frog Ringer's solution called ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.55) supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 µg/ml gentamicin. Current recordings were made 2-5 days later using the two-electrode voltage clamp technique with a Geneclamp 500 amplifier (Axon instruments, Foster City, CA) interfaced with a MacLab 2e chart recorder (ADI Instruments, Sydney, Australia) using the Chart software and a Digidata 1200 (Axon Instruments), which is controlled by an IBM-compatible computer installed with pClamp software, version 7.0 (Axon Instruments).

The current-voltage relationships for substrate-elicited conductances were determined by subtraction of steady-state current measurements in the absence of substrate obtained during 200-ms voltage pulses from 30 mV to potentials between 100 and +40 mV in 10 mV steps from corresponding current measurements in the presence of substrate. Current voltage measurements were normalized to the current generated by L-glutamate at 100 mV unless otherwise stated. In recordings in which the extracellular Cl concentration was altered, equimolar substitutions were made with bromide (Br), iodide (I), or nitrate (N O), and recordings were made with the bath grounded via a 3 M KCl/agar bridge connected to a 3 M KCl reservoir to minimize offset potentials. To measure reverse transport conductances, ND96 buffer was replaced with a high K⁺ buffer (46 mM NaCl, 50 mM KCl), and steady-state currents measured in the presence of 300 µM TBOA during 200-ms voltage pulses from 30 mV to potentials between 100 and +40 mV in 10 mV steps were subtracted from corresponding current measurements in high K⁺ buffer alone.

Radiotracer Flux Experiments-- The uptake of L-[³H]glutamate (Amersham Biosciences) was measured in oocytes expressing CLE1, V452C, and uninjected oocytes by placing five oocytes in ND96 buffer containing 10 µM L-[³H]glutamate. MTSET-treated cells were incubated in 1 mM MTSET for 5 min and washed three times in ND96 at room temperature before uptake was performed. After 5 min, L-[³H]glutamate uptake was terminated by three rapid washes in ice-cold ND96 buffer followed by lysis in 50 mM NaOH and scintillation counting.

The effects of MTSET treatment of V452C and CLE1 on D-[³H]aspartate (Amersham Biosciences) reverse transport were also measured. Oocytes were incubated with 10 µM D-[³H]aspartate in ND96 for 1 h at room temperature to load the oocytes with D-[³H]aspartate followed by washing the cells three times in ice-cold buffer. D-[³H]Aspartate-loaded cells were incubated in the presence or absence of 1 mM MTSET for 5 min at room temperature and washed three times in buffer before release was initiated. Single oocytes were placed in 500 µl of high K⁺ buffer (46 mM NaCl, 50 mM KCl) for 90 s, and 400 µl of the external solution was then removed for scintillation counting and measurement of the extent of reverse transport. Background rates of release were measured in the presence of 1 mM TBOA. Oocytes were also removed, and the level of radioactivity was measured to confirm the extent of loading of D-[³H]aspartate.

Analysis of Kinetic Data-- An analysis of kinetic data was carried out using the Kaleidagraph Software version 3.1. L-Glutamate dose responses were fitted by least squares as a function of current (I) to I/I_max = [S]/(EC₅₀ + [S]), where I_max is the maximal current, EC₅₀ is the concentration of L-glutamate that generates half-maximal current, and [S] is the L-glutamate concentration. TBOA is a competitive blocker of glutamate transport (17), and the following equation was used to estimate the IC₅₀ for TBOA, I/I_glu = 1  ([TBOA]/[TBOA + IC₅₀]). All values presented are the means of at least three cells ± S.E. One-way ANOVA test with the Bonferroni's post hoc test or the Student's t test were performed using GraphPad Prism (18) to assess the difference in the means. p < 0.05 were taken to be significant and are indicated in figures with an asterisk.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The application of MTS derivatives to the wild-type EAAT-1 transporter does not alter its transport activity (19). However, to ensure that further mutations do not expose a previously unreactive cysteine residue, a cysteine-less transporter was constructed. Conservative substitutions for the three endogenous cysteine residues (C186A, C252A, and C375A) resulted in a transporter termed CLE1. The application of L-glutamate or D-aspartate to oocytes expressing CLE1 elicits a conductance that is qualitatively similar to wild type in terms of current amplitude, rectification, and apparent substrate affinity (Fig. 1, A and C). The application of 1 mM MTSET for 5 min caused no change in the L-glutamate-elicited or D-aspartate-elicited conductances (Fig. 1, A and C).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   MTSET changes the substrate-activated conductance of V452C but not of CLE1. Current voltage plots of substrate-elicited conductances of CLE1 and V452C were measured in the presence of 100 µM L-glutamate (A and B) or 100 µM D-aspartate (C and D) before () and after () 5-min incubation with 1 mM MTSET (I(100 µM substrate in buffer)  I(buffer)). The data represented are normalized to the current elicited by 100 µM L-glutamate at 100 mV and represent the mean currents ± S.E. from five or more cells. After MTSET treatment of the V452C mutant transporter, the reversal potential of the L-glutamate-activated conductance shifted from +19.3 ± 1.7 mV to 18 ± 0.9 mV (n = 6). Similarly, D-aspartate-activated conductances were shifted from 1.3 ± 1.6 mV to 18 ± 0.9 mV (n = 6). The Student's t test showed that these reversal potential shifts were significant (p < 0.0001).

The application of 100 µM L-glutamate to oocytes expressing CLE1 or the V452C mutant generates similar conductances, which suggests that the cysteine mutation does not alter the structure or function of the transporter. The application of 1 mM MTSET does not affect the L-glutamate-activated conductance of CLE1 but does cause significant changes in the L-glutamate-activated conductance of the V452C mutant (Fig. 1B). The amplitude of the current is decreased at negative potentials, and the reversal potential of the current is shifted 40 mV to more negative membrane potentials to 18.7 ± 0.9 mV (n = 6, p < 0.0001). Similar reductions in current amplitude and reversal potential measurements are also observed for D-aspartate-activated conductances (Fig. 1D). Despite these differences, the EC₅₀ for the L-glutamate-activated conductances of the V452C mutant were not significantly different before and after treatment with MTSET or compared with the CLE1 transporter (Fig. 2) and the wild-type transporter (4, 20-22).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   MTSET does not change the apparent affinity of L-glutamate for CLE1 or V452C. L-Glutamate dose responses were measured for CLE1 () and V452C both before () and after () 1 mM MTSET application for 5 min with apparent affinity values for CLE1 (31 ± 7.8 µM) and for V452C before MTSET (20.5 ± 3.9 µM) and after MTSET treatment (23.7 ± 1.0 µM). Current measurements at 60 mV were normalized to the maximal current, and the data represent the mean ± S.E. from five cells for each condition. One-way ANOVA test with the Bonferroni's post hoc test showed no significance between the apparent affinity values.

In wild-type EAAT-1, the glutamate-activated conductance is composed of an inwardly rectifying glutamate transport conductance and an uncoupled chloride conductance (4, 22). In Xenopus laevis oocytes, the reversal potential for chloride ions is ~20 mV (3, 4, 23), which is similar to the reversal potential observed for the V452C mutant after MTSET treatment (18.7 ± 0.9 mV). Thus, the effect of MTSET may be to selectively reduce the transport component of the conductance with chloride contributing a greater proportion of the current, resulting in the current reversing the direction closer to the reversal potential for chloride ions. To confirm this hypothesis, L-[³H]glutamate uptake by oocytes expressing CLE1 and the V452C mutant with and without MTSET treatment was measured. Under control conditions with no MTSET treatment, the rates of L-[³H]glutamate uptake by oocytes expressing CLE1 and V452C-expressing oocytes are similar (Fig. 3). After incubation of the oocytes with 1 mM MTSET, the rate of L-[³H]glutamate uptake by oocytes expressing CLE1 is unaffected, whereas the rate of uptake by oocytes expressing the V452C mutant is reduced to background levels (Fig. 3). These results confirm that the rate of glutamate transport is unaffected by the V452C mutation and that application of MTSET to the V452C mutant transporter selectively inhibits the transport component of the glutamate-activated conductance.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   MTSET inhibits L-[3H]glutamate uptake via V452C. Uninjected (uninj.) oocytes and oocytes expressing CLE1 or V452C were incubated at room temperature in ND96 buffer containing 10 µM L-[3H]glutamate. Under control conditions (i.e. no incubation with MTSET), the uptake levels of CLE1 and V452C were similar (open bars). After a 5-min incubation with 1 mM MTSET prior to 10 µM L-[3H]glutamate addition, the uptake via CLE1 was unaffected, whereas the levels of uptake of the V452C mutant (closed bars) were significantly reduced (p < 0.001) compared with the uptake under control conditions (one-way ANOVA test with Bonferroni's post hoc test). Data represent mean uptake rates ± S.E. from five cells for each condition.

The uncoupled anion conductance of the glutamate transporter family displays a chaotropic selectivity sequence in which some anions are more permeant than others (4, 5, 22). With the more permeant anions in the extracellular solution, the substrate-induced currents are larger in amplitude and the reversal potential of these currents shifts to more hyperpolarized potentials as the proportion of the current being carried by the anions increases. When extracellular Cl is substituted with either bromide (Br), iodide (I), or nitrate (NO), the amplitude and reversal potential of the glutamate-activated currents of CLE1 and V452C (Fig. 4) are similar to that previously described for wild-type EAAT-1 (4, 22). MTSET treatment does not affect the amplitudes or reversal potentials of the glutamate-activated conductances of CLE1 but does cause significant changes in the V452C mutant (Fig. 4). MTSET treatment of V452C transporters increases the amplitude of the anion conductance and shifts the reversal potential to more hyperpolarized potentials. These results confirm that the application of glutamate to the mutant transporter after MTSET treatment still activates an anion conductance, but they also confirm that the nature of the anion conductance has not been altered.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   MTSET increases the uncoupled anion conductance of V452C. L-Glutamate elicited were measured as in Fig. 1 in frog Ringer phosphate buffer containing 96 mM NaCl, 96 mM NaBr, 96 mM NaI, or 96 mM NaNO3. Current-voltage relationships were measured before () and after a 5-min incubation () with 1 mM MTSET. The data are normalized to the current elicited by 100 µM L-glutamate in 96 mM NaCl at 100 mV and represent the mean currents ± S.E. from five cells.

The results presented thus far suggest that MTSET modification of the V452C mutant EAAT-1 does not affect glutamate binding and activation of the anion conductance but does inhibit glutamate transport. We have also investigated whether reverse transport and the reverse transport-activated chloride conductance (5) are similarly affected. To isolate reverse transport and reverse transport-activated chloride conductances, it was necessary to use the glutamate transport blocker TBOA (17). TBOA is a non-transportable competitive inhibitor of glutamate transport that binds to the extracellular-facing glutamate binding site. However, before using TBOA to characterize reverse transport, we investigated its effect on forward transport under standard ionic conditions to confirm that it still binds to the mutant transporter. After MTSET treatment, increasing doses of TBOA caused a progressive reduction in the glutamate-activated conductance with an IC₅₀ of 43 µM for CLE1 and 181 µM for the V452C transporter (n = 2, data not shown). Thus, although glutamate binding to either CLE1 or V452C is unaffected by MTSET treatment (see above), the affinity of TBOA for the MTSET-modified mutant is reduced. Nonetheless, high doses of TBOA used in the following experiment still block a large proportion of the conductance elicited by 30 µM L-glutamate.

Under conditions of elevated extracellular K⁺ and reduced extracellular Na⁺, glutamate transporters may function in reverse (1, 24). In X. laevis oocytes, this procedure may lead to the activation of a variety of conductances, but with the use of TBOA, it is possible to isolate the conductance changes that are specific to reverse operation of glutamate transporters. Under conditions of elevated extracellular K⁺ (50 mM) and reduced extracellular Na⁺ (46 mM, the application of 300 µM TBOA blocks conductances because of reverse transport in both CLE1 and the mutant transporter V452C, which reverse the direction at 48 ± 5 mV (n = 3) and 52 ± 1 mV (n = 3), respectively, and are ~10% of the forward transport conductance activated by 30 µM L-glutamate (Fig. 5, A and B). After MTSET treatment, TBOA block of the reverse transport conductance of CLE1 is unaffected. However, high extracellular K⁺ does not activate any conductance of the MTSET-modified mutant V452C, which can be blocked by TBOA. The conductance activated by reverse transport in the wild-type transporter will also have two components comprised of the coupled movement of glutamate/3Na⁺/H⁺/K⁺, and the reverse transport-activated chloride conductance (5, 22, 25). The lack of any conductance blocked by TBOA after MTSET treatment of V452C suggests that both the reverse transport and reverse transport-activated chloride conductance are not activated by high extracellular K⁺.

View larger version (9K): [in this window] [in a new window]   Fig. 5.   MTSET blocks reverse glutamate transport and the reverse transport-activated anion conductance. TBOA (300 µM) blocks the reverse transport currents of CLE1 (A) and V452C (B) () (see "Materials and Methods"). After application of 1 mM MTSET for 5 min (), TBOA blocks the reverse transport conductance via CLE1 (A) but does not block any reverse transport conductances via V452C (B). A and B, the currents plotted are (I(buffer)  I(300 µM TBOA in buffer)), and the values normalized to the currents measured for 30 µM L-glutamate at 100 mV for each cell. Data represent mean values ± S.E. from three cells for each condition. C, reverse transport of D-[3H]aspartate from pre-loaded oocytes (see "Materials and Methods") was significantly reduced (p < 0.05) after MTSET treatment of V452C. Transporter-specific release of D-[3H]aspartate was calculated by subtraction of the background level of release, which was estimated by incubating oocytes-expressing V452C in high K+ buffer containing 1 mM TBOA. Data represent mean values ± S.E. from five cells for each condition. Significant differences were demonstrated using the unpaired Student's t test. Similar experiments were also carried out for oocytes expressing CLE1, but no significant differences in the rates of reverse transport with and without MTSET treatment were observed.

To confirm these findings, the reverse transport of D-[³H]aspartate was measured. Oocytes expressing CLE1 or V452C oocytes were preloaded with D-[³H]aspartate, which was chosen as it is a non-metabolized substrate of EAAT-1. Control oocytes were placed in ND96 at room temperature, whereas MTSET-treated cells were incubated with 1 mM MTSET for 5 min. Oocytes were washed and then placed in the same high K⁺ buffer used for the electrophysiological recordings. Transporter-specific release of D-[³H]aspartate was determined by the subtraction of the amount of release measured in the presence of 1 mM TBOA. MTSET treatment significantly reduced the amount of V452C-specific release to background levels (Fig. 5C) but did not affect the amount of CLE1-specific D-[³H]aspartate release (data not shown). The lack of D-[³H]aspartate release and the lack of any conductance blocked by TBOA after MTSET treatment of V452C suggest that both the reverse transport and reverse transport-activated chloride conductance are prevented by the MTSET treatment. These results are in contrast to that observed for the effects of MTSET on forward transport where only the coupled transport conductance was inhibited, and these results suggest that the effects of MTSET are asymmetrical with respect to forward and reverse transport activation of the chloride conductance.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The observation that glutamate transporters may have a secondary function as glutamate-activated anion channels (3, 26, 27) has raised a number of questions. 1) What is the structural basis for the different functions? 2) How are these two processes coordinated? 3) Is it possible to independently modulate the two processes? In this study, we have described a mutant EAAT-1 transporter V452C that when modified with MTSET selectively blocks glutamate transport while retaining the glutamate-activated uncoupled chloride channel function of the transporter. The EC₅₀ for glutamate activation of the anion conductance of the MTSET-modified transporter is not significantly different from the EC₅₀ for glutamate transport and anion conductance activation of the CLE1 or wild-type EAAT-1, which demonstrates that the glutamate recognition site is not significantly altered in the MTSET-modified mutant transporter. Valine 452 is located in a hydrophobic "linker" region that is exposed to the extracellular solution and lies between the reentrant loop that is likely to form the glutamate recognition site (12, 19, 20, 28-32) and is a putative -helical transmembrane domain containing an arginine residue that may interact with the -carboxyl group of glutamate (33). The results presented in this study confirm that this linker region is unlikely to be directly involved in glutamate recognition and activation of the anion conductance but do suggest that conformational changes mediated by this region may be required for the translocation of glutamate through the transporter.

The activation of the anion conductance of the MTSET-modified V452C mutant transporter requires glutamate binding but clearly does not require active transport, which confirms previous studies (5, 22) suggesting that the binding of glutamate to the transporter without transport is sufficient for activation of the anion conductance. The anion conductance of the MTSET-modified mutant transporter shows some subtle differences compared with the anion conductance that is activated by glutamate binding/transport of the wild-type EAAT-1 and the CLE1 transporter. In addition to the shift in reversal potential of the glutamate-activated conductance before and after MTSET treatment because of the removal of the transport component of the conductance, the amplitude of the anion conductance is significantly greater. These changes in anion conductance amplitude are most apparent with the more permeant anions iodide and nitrate, and in the case of nitrate, the slope conductance measured between 0 and +40 mV was increased 1.9-fold after MTSET treatment. The simplest explanation for these differences in the anion conductance properties is that the MTSET treatment prevents the transporter from undergoing conformational changes required for transport and increases either the open time for the anion channel or the frequency of anion channel opening resulting in a larger anion conductance.

The results presented suggest that MTSET modification of V452C locks the transporter into a conformation that allows glutamate binding and activation of the anion conductance but does not allow translocation of glutamate through the transporter. However, under the conditions that cause the wild-type and CLE1 transporters to function in reverse, MTSET modification of the mutant appears to prevent both reverse transport and reverse transport activation of the anion conductance. This observation can be explained by postulating that MTSET modification of the mutant transporter locks the transporter into a conformation that allows glutamate to bind to an outward facing recognition site but does not allow reorientation of the transporter to bind glutamate from the intracellular surface. Thus, in the absence of glutamate binding to the intracellular recognition site, there is no anion conductance activated and no transporter-associated conductances for TBOA to block (Fig. 5, B and C).

The selective manipulation of the transport properties of EAAT-1 without affecting the anion conductance is in contrast to the effects of Zn²⁺ on the glutamate transporter EAAT-4 in which Zn²⁺ selectively inhibits the anion conductance without affecting transport (13). These two studies (34, 35) clearly demonstrate that it is possible to selectively manipulate the two functions of glutamate transporters, which highlights the possibility that the two functions are mediated via separate pore structures. The quaternary structure of the glutamate transporters is unknown, but two studies have suggested that that the transporters associate to form homomultimeric complexes. Electron micrograph studies of EAAT-3 expressed in oocyte membranes suggest that the transporters exist as a pentameric assembly (35). It was suggested that each transporter "subunit" conducted coupled glutamate transport, whereas the association of the subunits allows the formation of a central chloride channel. If this model is correct, MTSET modification of the mutant transporter may prevent translocation of glutamate by each of the subunits but not alter the conformational changes required for the opening of the central chloride ion channel. Although the structural model proposed by Eskandari et al. (35) can explain how it is possible to independently manipulate the transport and anion channel functions of the transporter, the results presented in this study can also be equally well explained by a common pathway for transport and chloride ion permeation in which the transporter is able to switch between the two modes of operation. Nevertheless, our results do suggest that the transport and chloride channel properties are mediated by different conformational states of the transporter, and additional site-directed mutagenesis studies, which identify distinct molecular determinants of the two functions, will be required to distinguish between the single and multiple pore models for glutamate transporter function.

Since the original submission of this paper, a similar study has been published by Seal et al. (36). This study investigated the effects of MTS reagents on a valine residue 449 to cysteine mutation in EAAT-1 (36) and also showed that MTS reagents abolish glutamate transport but do not affect the anion conductance. If the domain containing these residues is an -helix, the two residues (449 and 452) would be located on the same side of the helix and suggests that this domain may form part of a structure necessary for the transduction of substrate binding into substrate translocation through the transporter.

	ACKNOWLEDGEMENTS

We thank Dr. Hue Tran and Kong Li for the maintenance of the X. laevis colony and the isolation and preparation of oocytes.

	FOOTNOTES

^* This work was supported by the Australian National Health and Medical Research Council.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.

Supported by an Australian postgraduate award.

^§ To whom correspondence should be addressed. Tel.: 61-2-9351-6734; Fax: 61-2-9351-3868; E-mail: robv@pharmacol.usyd.edu.au.

Published, JBC Papers in Press, January 28, 2002, DOI 10.1074/jbc.M109970200

	ABBREVIATIONS

The abbreviations used are: EAATs, excitatory amino acid transporters; CLE1, cysteine-less EAAT-1; V452C, CLE1 mutant V452C; MTS, methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate; TBOA, DL-threo--benzyloxyaspartate; ANOVA, analysis of variance.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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