Two conformational changes are associated with glutamate translocation by the glutamate transporter EAAC1

Glutamate is transported across membranes by means of a carrier mechanism that is thought to require conformational changes of the transport protein. In this work, we have determined the thermodynamic parameters of glutamate and the Na+ binding steps to their extracellular binding sites along with the activation parameters of rapid, glutamate-induced processes in the transport cycle by analyzing the temperature dependence of glutamate transport at steady state and pre-steady state. Our results suggest that glutamate binding to the transporter is driven by a negative reaction enthalpy (DeltaH0 = -33 kJ/mol), whereas the tighter binding of the non-transportable inhibitor TBOA is caused by an additional increase in entropy. Processes linked to the binding of glutamate and Na+ to the transporter are associated with low activation barriers, indicative of diffusion-controlled reactions. The activation enthalpies of two processes in the glutamate translocation branch of the transport cycle were DeltaH++ = 95 kJ/mol and DeltaH++ = 120 kJ/mol, respectively. Such large values of DeltaH++ suggest that these processes are rate-limited by conformational changes of the transporter. We also found a large activation barrier for steady-state glutamate transport, which is rate-limited by the K+-dependent relocation of the empty transporter. Together, these results suggest that two conformational changes accompany glutamate translocation and at least one conformational change accompanies the relocation of the empty transporter. We interpret the data with an alternating access model that includes the closing and opening of an extracellular and an intracellular gate, respectively, in analogy to a hypothetical model proposed previously on the basis of the crystal structure of the bacterial glutamate transporter GltPh.     

Effect of benzodiazepines on the epithelial and neuronal high-affinity glutamate transporter EAAC1

EAAC1-mediated glutamate transport concentrates glutamate across plasma membranes of brain neurons and epithelia. In brain, EAAC1 provides a presynaptic uptake mechanism to terminate the excitatory action of released glutamate and to keep its extracellular concentration below toxic levels. Here we report the effect of well known anxiolytic compounds, benzodiazepines, on glutamate transport in EAAC1-stably transfected Chinese hamster ovary (CHO) cells and in EAAC1-expressing Xenopus laevis oocytes. Functional properties of EAAC1 agreed well with already reported characteristics of the neuronal high-affinity glutamate transporter (Km D-Asp,CHO cells: 2.23+/-0.15 microM; Km D-Asp,oocytes: 17.01+/-3.42 microM). In both expression systems, low drug concentrations (10-100 microM) activated substrate uptake (up to 200% of control), whereas concentrations in the millimolar range inhibited (up to 50%). Furthermore, the activation was more pronounced at low substrate concentrations (1 microM), and the inhibition was attenuated. The activity of other sodium cotransporters such as the sodium/D-glucose cotransporter SGLT1, stably transfected in CHO cells, was not affected by benzodiazepines. In electrophysiological studies, these drugs also failed to change the membrane potential of EAAC1-expressing Xenopus laevis oocytes. These results suggest a direct action on the glutamate transporter itself without modifying the general driving forces. Thus, in vivo low concentrations of benzodiazepines may reduce synaptic glutamate concentrations by increased uptake, providing an additional mechanism to modulate neuronal excitability.     

Zn(2+) inhibits the anion conductance of the glutamate transporter EEAT4

Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na(+) ions and 1 H(+) and the counter-transport of 1 K(+) ion, which ensures that extracellular glutamate concentrations are maintained in the submicromolar range. In addition to the coupled ion fluxes, glutamate transport activates an uncoupled anion conductance that does not influence the rate or direction of transport but may have the capacity to influence the excitability of the cell. Free Zn(2+) ions are often co-localized with glutamate in the central nervous system and have the capacity to modulate the dynamics of excitatory neurotransmission. In this study we demonstrate that Zn(2+) ions inhibit the uncoupled anion conductance and also reduce the affinity of L-aspartate for EAAT4. The molecular basis for this effect was investigated using site-directed mutagenesis. Two histidine residues in the extracellular loop between transmembrane domains three and four of EAAT4 appear to confer Zn(2+) inhibition of the anion conductance.     

Individual subunits of the glutamate transporter EAAC1 homotrimer function independently of each other

Glutamate transporters are thought to be assembled as trimers of identical subunits that line a central hole, possibly the permeation pathway for anions. Here, we have tested the effect of multimerization on the transporter function. To do so, we coexpressed EAAC1(WT) with the mutant transporter EAAC1(R446Q), which transports glutamine but not glutamate. Application of 50 microM glutamate or 50 microM glutamine to cells coexpressing similar numbers of both transporters resulted in anion currents of 165 and 130 pA, respectively. Application of both substrates at the same time generated an anion current of 297 pA, demonstrating that the currents catalyzed by the wild-type and mutant transporter subunits are purely additive. This result is unexpected for anion permeation through a central pore but could be explained by anion permeation through independently functioning subunits. To further test the subunit independence, we coexpressed EAAC1(WT) and EAAC1(H295K), a transporter with a 90-fold reduced glutamate affinity as compared to EAAC1(WT), and determined the glutamate concentration dependence of currents of the mixed transporter population. The data were consistent with two independent populations of transporters with apparent glutamate affinities similar to those of EAAC1(H295K) and EAAC1(WT), respectively. Finally, we coexpressed EAAC1(WT) with the pH-independent mutant transporter EAAC1(E373Q), showing two independent populations of transporters, one being pH-dependent and the other being pH-independent. In conclusion, we propose that EAAC1 assembles as trimers of identical subunits but that the individual subunits in the trimer function independently of each other.     

Distinct conformational states mediate the transport and anion channel properties of the glutamate transporter EAAT-1

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.     

Modulation of the neural glutamate transporter EAAC1 by the addicsin-interacting protein ARL6IP1

Addicsin (Arl6ip5) is a murine homologue of rat glutamate transporter-associated protein 3-18 (GTRAP3-18), a putative negative modulator of Na+-dependent neural glutamate transporter-excitatory amino acid carrier 1 (EAAC1). Here we report that ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1) is a novel addicsin-associated partner that indirectly promotes EAAC1-mediated glutamate transport activity in a protein kinase C activity-dependent manner. Like addicsin, Arl6ip1 is expressed in numerous tissues and proved likely to be co-localized with addicsin in certain neurons in the matured brain. Arl6ip1 was not translocated from the subcellular compartments under any of the test conditions and had no association with any molecules on the plasma membrane. Immunoprecipitation assay demonstrated that Arl6ip1 bound directly to addicsin and that the hydrophobic region located at amino acids 103-117 of addicsin was crucial to the formation of the Arl6ip1-addicsin heterodimer and addicsin homodimer. Glutamate transport assay revealed that increasing the expression of Arl6ip1 in C6BU-1 cells markedly enhanced Na+-dependent EAAC1-mediated glutamate transport activity in the presence of 100 nm phorbol 12-myristate 13-acetate. Under these conditions, kinetic analyses demonstrated that EAAC1 altered glutamate transport activity by increasing its glutamate affinity but not its maximal velocity. Meanwhile, increasing expression of addicsin Y110A/L112A mutant lacking binding ability for Arl6ip1 showed no enhancement of EAAC1-mediated glutamate transport activity, regardless of phorbol 12-myristate 13-acetate activation, suggesting that association between addicsin and Arl6ip1 causes altered EAAC1-mediated glutamate transport activity. Our findings suggest that Arl6ip1 is a novel addicsin-associated partner that promotes EAAC1-mediated glutamate transport activity by decreasing the number of addicsin molecules available for interaction with EAAC1.     

The conserved histidine 295 does not contribute to proton cotransport by the glutamate transporter EAAC1

Transmembrane glutamate transport by the excitatory amino acid carrier (EAAC1) is coupled to the cotransport of three Na(+) ions and one proton. Previously, we suggested that the mechanism of H(+) cotransport involves protonation of the conserved glutamate residue E373. However, it was also speculated that the cotransported proton is shared in a H(+)-binding network, possibly involving the conserved histidine 295 in the sixth transmembrane domain of EAAC1. Here, we used site-directed mutagenesis together with pre-steady-state electrophysiological analysis of the mutant transporters to test the protonation state of H295 and to determine its involvement in proton transport by EAAC1. Our results show that replacement of H295 with glutamine, an amino acid residue that cannot be protonated, generates a fully functional transporter with transport kinetics that are close to those of the wild-type EAAC1. In contrast, replacement with lysine results in a transporter in which substrate binding and translocation are dramatically inhibited. Furthermore, it is demonstrated that the effect of the histidine 295 to lysine mutation on the glutamate affinity is caused by its positive charge, since wild-type-like affinity can be restored by changing the extracellular pH to 10.0, thus partially deprotonating H295K. Together, these results suggest that histidine 295 is not protonated in EAAC1 at physiological pH and, thus, does not contribute to H(+) cotransport. This conclusion is supported by data from H295C-E373C double mutant transporters which demonstrate that these residues cannot be linked by oxidation, indicating that H295 and E373 are not close in space and do not form a proton binding network. A kinetic scheme is used to quantify the results, which includes binding of the cotransported proton to E373 and binding of a modulatory, nontransported proton to the amino acid side chain in position 295.     

Reticulon RTN2B regulates trafficking and function of neuronal glutamate transporter EAAC1

Excitatory amino acid transporters (EAATs) are the primary regulators of extracellular glutamate concentrations in the central nervous system. Their dysfunction may contribute to several neurological diseases. To date, five distinct mammalian glutamate transporters have been cloned. In brain, EAAC1 (excitatory amino acid carrier 1) is the primary neuronal glutamate transporter, localized on the perisynaptic membranes that are near release sites. Despite its potential importance in synaptic actions, little is known concerning the regulation of EAAC1 trafficking from the endoplasmic reticulum (ER) to the cell surface. Previously, we identified an EAAC1-associated protein, GTRAP3-18, an ER protein that prevents ER exit of EAAC1 when induced. Here we show that RTN2B, a member of the reticulon protein family that mainly localizes in the ER and ER exit sites interacts with EAAC1 and GTRAP3-18. EAAC1 and GTRAP3-18 bind to different regions of RTN2B. Each protein can separately and independently form complexes with EAAC1. RTN2B enhances ER exit and the cell surface composition of EAAC1 in heterologous cells. Expression of short interfering RNA-mediated knockdown of RTN2B decreases the EAAC1 protein level in neurons. Overall, our results suggest that RTN2B functions as a positive regulator in the delivery of EAAC1 from the ER to the cell surface. These studies indicate that transporter exit from the ER controlled by the interaction with its ER binding partner represents a critical regulatory step in glutamate transporter trafficking to the cell surface.     

Localization and possible function of the glutamate transporter,EAAC1, in the rat retina

We investigated the localization and possible function of EAAC1 in the rat retina. Immunocytochemical localization of EAAC1 at the light-microscopic level revealed a fine dust-like labelling pattern across the two synaptic layers. Horizontal cell and subpopulations of amacrine cell somata were labelled, as were some somata within the ganglion cell layer. Some immunoreactive puncta were observed within the cytoplasm of amacrine cells, in regions well away from synaptic sites. At the ultrastructural level, EAAC1 immunolabelled one postsynaptic element at synapses and also processes well away from the synaptic release site. Since EAAC1 was localized away from synaptic sites, we evaluated the role EAAC1 plays in GABA formation by measuring GABA concentrations via reversed-phase high-performance liquid chromatography following incubation of retinae in enzyme and glutamate uptake inhibitors. Incubation of retinae in D-threo-beta-hydroxyaspartate or D/ L-threo-beta-benzyloxyaspartate, which are known to inhibit the glutamate transporters GLAST1, GLT1, and EAAC1, caused a decrease in GABA synthesis by around 50%. Incubation in 6-diazo-5-oxo- L-norleucine, a phosphate-activated glutaminase inhibitor, decreased GABA formation by 40%. Taken together with the anatomical data, the results of this study suggest that EAAC1 plays very little role in GABA synthesis - indeed GABA formation occurs predominantly from glutamine. By virtue of its location both near and well away from synaptic release sites, EAAC1 may regulate glutamate uptake differentially.     

The uncoupled chloride conductance of a bacterial glutamate transporter homolog

Glutamate transporters (EAATs) are pivotal in mammalian synaptic transmission, tightly regulating synaptic levels of this excitatory neurotransmitter. In addition to coupled glutamate transport, the EAATs also show an uncoupled Cl(-) conductance, whose physiological importance has recently been demonstrated. Little is yet known about the molecular mechanism of chloride permeation. Here we show that Glt(Ph), a bacterial EAAT homolog whose structure has been determined, displays an uncoupled Cl(-) conductance that can determine the rate of substrate uptake. A mutation analogous to one known to specifically affect Cl(-) movement in EAAT1 has similar effects on Glt(Ph), suggesting that this protein is an excellent structural model for understanding Cl(-) permeation through the EAATs. We also observed an uncoupled Cl(-) conductance in another bacterial EAAT homolog but not in a homolog of the Na(+)/Cl(-)-coupled neurotransmitter transporters.     

The discovery of slowness: low-capacity transport and slow anion channel gating by the glutamate transporter EAAT5

Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na⁺ and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na⁺ and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a K_m= 61 ± 11 μM. Binding of Na⁺ to the empty transporter is associated with a K_m = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a K_m = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na⁺ binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system.     

Different functional roles of arginine residues 39 and 61 and tyrosine residue 98 in transport and channel mode of the glutamate transporter EAAC1

The excitatory amino acid transporter EAAC1 is an electrogenic Na+ - and K+ -gradient-driven transporter. In addition, the transporter mediates in the presence of Na+ and glutamate an anion conductance uncoupled from the transport of the glutamate. The first two N-terminal domains, important for forming the conductance mode, are extracellularly bordered by positively charged arginine residues, R39 and R61, being completely conserved throughout the transporter family. Also the conserved tyrosine residue Y98 could be important for Cl- conductance. We have investigated, by measurements of glutamate uptake and glutamate-induced currents, the effects of mutation of the arginines and the tyrosine to alanine. The mutation R39A hardly affects transport and channel mode. The mutation R61A, on the other hand, reduces the activity of transport but stimulates the channel conductance. In addition, the apparent Km values for glutamate uptake and for the glutamate-activated current are reduced. Glutamate stimulation of current seems to be associated with a voltage-dependent step, and the apparent valence of charge moved during binding is reduced in the R61A mutant. The mutation Y98A leads to reduced function with reduced apparent Km value for glutamate, and with strong reduction of the selectivity ration between NO3- and Cl- of the conductance mode.     

Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol

A co-ordinated regulation between neurons and astrocytes is essential for the control of extracellular glutamate concentration. Here, we have investigated the influence of astrocytes and glia-derived cholesterol on the regulation of glutamate transport in primary neuronal cultures from rat embryonic cortices. Glutamate uptake rate and expression of the neuronal glutamate transporter EAAC1 were low when neurons were grown without astrocytes and neurons were unable to clear extracellular glutamate. Treatment of the neuronal cultures with glial conditioned medium (GCM) increased glutamate uptake Vmax, EAAC1 expression and restored the capacity of neurons to eliminate extracellular glutamate. Thus, astrocytes up-regulate the activity and expression of EAAC1 in neurons. We further showed that cholesterol, present in GCM, increased glutamate uptake activity when added directly to neurons and had no effect on glutamate transporter expression. Furthermore, part of the GCM-induced effect on glutamate transport activity was lost when cholesterol was removed from GCM (low cholesterol-GCM) and was restored when cholesterol was added to low cholesterol-GCM. This demonstrates that glia-derived cholesterol regulates glutamate transport activity. With these experiments, we provide new evidences for neuronal glutamate transport regulation by astrocytes and identified cholesterol as one of the factors implicated in this regulation.     

A carboxyl-terminal determinant of the neuronal glutamate transporter, EAAC1, is required for platelet-derived growth factor-dependent trafficking

The neuronal glutamate transporter, EAAC1 (excitatory amino acid carrier 1), undergoes rapid regulation after treatment with platelet-derived growth factor (PDGF) or phorbol ester in C6 glioma cells and neurons. A large intracellular pool of EAAC1 exists, from which transporters are redistributed to the cell surface in response to these signals. Here we show that PDGF had no effect on subcellular localization of the glial glutamate transporter, GLT-1, after transfection into C6 glioma cells. Chimeras consisting of domains from EAAC1 or GLT-1 were used to investigate structural motifs involved in PDGF-dependent redistribution of EAAC1. PDGF did not induce trafficking of an EAAC1 chimera containing the carboxyl-terminal domain of GLT-1; however, it did induce trafficking of a GLT-1 chimera containing the carboxyl-terminal domain of EAAC1. A truncated mutant of EAAC1 lacking 10 carboxyl-terminal amino acids was responsive to PDGF, whereas a mutant lacking 20 residues was not. Alanine substitution mutagenesis in this region revealed a short motif, (502)YVN(504), necessary for regulated trafficking. This motif was also involved in protein kinase C-dependent trafficking, as mutant transporters exhibited an attenuated response to phorbol ester. Interestingly, the presence of YVN in the homologous region of a nonresponsive chimera was not sufficient to confer regulated trafficking; however, the presence of a 12-amino acid motif starting at this Tyr residue was sufficient to confer responsiveness to PDGF. These studies identify a novel motif within the carboxyl terminus of EAAC1 which is required for regulated trafficking. The possibility that this motif targets EAAC1 to an intracellular, "regulated pool" is discussed.     

Zinc potentiates an uncoupled anion conductance associated with the dopamine transporter

Binding of Zn(2+) to an endogenous binding site in the dopamine transporter (DAT) leads to inhibition of dopamine (DA) uptake and enhancement of carrier-mediated substrate efflux. To elucidate the molecular mechanism for this dual effect, we expressed the DAT and selected mutants in Xenopus laevis oocytes and applied the two-electrode voltage clamp technique together with substrate flux studies employing radiolabeled tracers. Under voltage clamp conditions we found that Zn(2+) (10 mum) enhanced the current induced by both DA and amphetamine. This was not accompanied by a change in the uptake rate but by a marked increase in the charge/DA flux coupling ratio as assessed from concomitant measurements of [(3)H]DA uptake and currents in voltage-clamped oocytes. These data suggest that Zn(2+) facilitates an uncoupled ion conductance mediated by DAT. Whereas this required substrate in the wild type (WT), we observed that Zn(2+) by itself activated such a conductance in a previously described mutant (Y335A). This signifies that the conductance is not strictly dependent on an active transport process. Ion substitution experiments in Y335A, as well as in WT, indicated that the uncoupled conductance activated by Zn(2+) was mainly carried by Cl(-). Experiments in oocytes under non-voltage-clamped conditions revealed furthermore that Zn(2+) could enhance the depolarizing effect of substrates in oocytes expressing WT. The data suggest that by potentiating an uncoupled Cl(-) conductance, Zn(2+) is capable of modulating the membrane potential of cells expressing DAT and as a result cause simultaneous inhibition of uptake and enhancement of efflux.     

Evidence for a second binding/transport site for chloride in erythrocyte anion transporter AE1 modified at glutamate 681

Transport kinetics have been examined in erythrocyte anion transporter AE1 that has been chemically modified to convert glutamate 681 to an alcohol (E681OH AE1). Outward conductive Cl(-) flux in E681OH AE1 is inhibited by removal of extracellular Cl(-); this effect is the opposite of that in native AE1 and is consistent with coupled electrogenic 2:1 Cl(-)/Cl(-) exchange. A second Cl(-) binding/transport site is also suggested by the characteristics of (35)SO(4)(2-) flux in E681OH AE1: bilateral and cis Cl(-), which are normally inhibitory, accelerate (35)SO(4)(2-) flux. These effects would be expected if Cl(-) binds to a second transport site on SO(4)(2-)-loaded E681OH AE1, thereby allowing Cl(-)/SO(4)(2-) cotransport. Alternatively, the data can be explained without proposing Cl(-)/SO(4)(2-) cotransport if the rate-limiting event for (35)SO(4)(2-)/SO(4)(2-) exchange is external SO(4)(2-) release, and the binding of external Cl(-) accelerates SO(4)(2-) release. With either interpretation, these data indicate that E681OH AE1 has a binding/transport site for Cl(-) that is distinct from the main transport site. The effects of graded modification of E681 or inhibition by H(2)DIDS are consistent with the idea that the new Cl(-) binding site is on the same E681OH-modified subunit of the AE1 dimer as the normal transport site.     

Biosynthesis,intracellular targeting,and degradation of the EAAC1 glutamate/aspartate transporter in C6 glioma cells

Rat C6 glioma cells were used as a model system to study the biosynthesis, intracellular targeting, and degradation of the EAAC1 transporter, a sodium-dependent glutamate/aspartate transport protein that encodes System X(-)A,G activity. At steady state, nearly 70% of the EAAC1 transporter was located at the cell surface. The newly synthesized EAAC1 protein was co-translationally N-glycosylated with high mannose oligosaccharide chains that were processed into complex-type sugar chains as the protein matured. The final maturation steps for EAAC1 protein coincided with its plasma membrane arrival, which was first detected at about 45 min after the initial synthesis. The newly synthesized EAAC1 protein was protected from degradation during the maturation and targeting process, as well as during the first 5 h after plasma membrane arrival. After this initial lag period, both the newly synthesized transporter and the total cellular EAAC1 pool were degraded by first order kinetics with a half-life of 6 h. These results represent the first analysis of the synthesis and degradation of the EAAC1 amino acid transporter.     

Cytoskeleton-related trafficking of the EAAC1 glutamate transporter after activation of the G(q/11)-coupled neurotensin receptor NTS1

The possible modulation of the glutamate transporter EAAC1 by a class A G protein-coupled receptor was studied in transfected C6 glioma cells stably expressing the high-affinity neurotensin receptor NTS1. Brief exposure (5 min) to neurotensin increased Na(+)-dependent D-[(3)H]aspartate uptake by about 70%. The effect of neurotensin was found to result from an increase in cell surface expression of EAAC1 and accordingly, cytochalasin D and colchicine were shown to block the effect of neurotensin on aspartate uptake, suggesting that the cytoskeleton participates in this regulation. Neither protein kinase C nor phosphatidylinositol 3-kinase activities, two intracellular signaling pathways known to modulate EAAC1, was required for EAAC1-mediated aspartate transport regulation by neurotensin. Together, these results provide evidence for an acute regulation of EAAC1 trafficking after activation of a G protein-coupled receptor.     

A dominant-negative variant of SNAP-23 decreases the cell surface expression of the neuronal glutamate transporter EAAC1 by slowing constitutive delivery

A family of high-affinity transporters controls the extracellular concentration of glutamate in the brain, ensuring appropriate excitatory signaling and preventing excitotoxicity. There is evidence that one of the neuronal glutamate transporters, EAAC1, is rapidly recycled on and off the plasma membrane with a half-life of no more than 5-7 min in both C6 glioma cells and cortical neurons. Syntaxin 1A has been implicated in the trafficking of several neurotransmitter transporters and in the regulation of EAAC1, but it has not been determined if this SNARE protein is required for EAAC1 trafficking. Expression of two different sets of SNARE proteins was examined in C6 glioma with Western blotting. These cells did not express syntaxin 1A, vesicle-associated membrane protein-1 (VAMP1), or synaptosomal-associated protein of 25 kDa (SNAP-25), but did express a family of SNARE proteins that has been implicated in glucose transporter trafficking, including syntaxin 4, vesicle-associated membrane protein-2 (VAMP2), and synaptosomal-associated protein of 23 kDa (SNAP-23). cDNAs encoding variants of SNAP-23 were co-transfected with Myc-tagged EAAC1 to determine if SNAP-23 function was required for maintenance of EAAC1 surface expression. Expression of a dominant-negative variant of SNAP-23 that lacks a domain required for SNARE complex assembly decreased the fraction of EAAC1 found on the cell surface and decreased total EAAC1 expression, while two control constructs had no effect. The dominant-negative variant of SNAP-23 also slowed the rate of EAAC1 delivery to the plasma membrane. These data strongly suggest that syntaxin 1A is not required for EAAC1 trafficking and provide evidence that SNAP-23 is required for constitutive recycling of EAAC1.