Glycine neurotransmitter transporters: an update

Glycine accomplishes several functions as a transmitter in the central nervous system(CNS). As an inhibitory neurotransmitter, it participates in the processing of motor and sensory information that permits movement, vision, and audition. This action of glycine is mediated by the strychnine-sensitive glycine receptor, whose activation produces inhibitory post-synaptic potentials. In some areas of the CNS, glycine seems to be co-released with GABA, the main inhibitory amino acid neurotransmitter. In addition, glycine modulates excitatory neurotransmission by potentiating the action of glutamate at N-methyl-D-aspartate (NMDA) receptors. It is believed that the termination of the different synaptic actions of glycine is produced by rapid reuptake through two sodium-and-chloride-coupled transporters, GLYT1 and GLYT2, located in the plasma membrane of glial cells or pre-synaptic terminals, respectively. Glycine transporters may become major targets for therapeutic of pathological alterations in synaptic function. This article reviews recent progress on the study of the molecular heterogeneity, localization, function, structure, regulation and pharmacology of the glycine transporter     

Molecular biology of glycinergic neurotransmission

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem of vertebrates. Glycine is accumulated into synaptic vesicles by a proton-coupled transport system and released to the synaptic cleft after depolarization of the presynaptic terminal. The inhibitory action of glycine is mediated by pentameric glycine receptors (GlyR) that belong to the ligand-gated ion channel superfamily. The synaptic action of glycine is terminated by two sodium- and chloride-coupled transporters, GLYT1 and GLYT2, located in the glial plasma membrane and in the presynaptic terminals, respectively. Dysfunction of inhibitory glycinergic neurotransmission is associated with several forms of inherited mammalian myoclonus. In addition, glycine could participate in excitatory neurotransmission by modulating the activity of the NMDA subtype of glutamate receptor. In this article, we discuss recent progress in our understanding of the molecular mechanisms that underlie the physiology and pathology of glycinergic neurotransmission.     

Molecular basis for substrate discrimination by glycine transporters

Glycine is an inhibitory neurotransmitter in the spinal cord and brain stem, where it acts on strychnine-sensitive glycine receptors, and is also an excitatory neurotransmitter throughout the brain and spinal cord, where it acts on the N-methyl-d-aspartate family of receptors. There are two Na(+)/Cl(-)-dependent glycine transporters, GLYT1 and GLYT2, which control extracellular glycine concentrations and these transporters show differences in substrate selectivity and blocker sensitivity. A bacterial Na(+)-dependent leucine transporter (LeuT(Aa)) has recently been crystallized and its structure determined. When the amino acid residues within the leucine binding site of LeuT(Aa) are aligned with residues of the two glycine transporters there are a number of identical residues and also some key differences. In this report, we demonstrate that the LeuT(Aa) structure represents a good working model of the Na(+)/Cl(-)-dependent neurotransmitters and that differences in substrate selectivity can be attributed to a single difference of a glycine residue in transmembrane domain 6 of GLYT1 for a serine residue at the corresponding position of GLYT2.     

Glycine transporters and synaptic function

Glycine is an inhibitory neurotransmitter that is mainly active in the caudal areas of the CNS. However, glycine also participates in excitatory neurotransmission since it is a co-agonist of the NMDA subtype of glutamate receptors. The concentration of glycine at synapses is mainly controlled by two sodium and chloride dependent transporters, GLYT1 and GLYT2, proteins that display a complementary distribution and activity in the nervous system. Our understanding of the physiological role of these transporters has advanced recently, thanks to the development of specific inhibitors and the generation of mice defective in the corresponding genes. In addition, the three-dimensional resolution of the structure of a bacterial homologue has shed light on the mechanisms of glycine transport. It is likely that this knowledge will prove to be useful for the development of drugs with antipsychotic, procognitive or analgesic properties.     

Molecular basis of the differential interaction with lithium of glycine transporters GLYT1 and GLYT2

Glycine synaptic levels are controlled by glycine transporters (GLYTs) catalyzing Na(+)/Cl(-)/glycine cotransport. GLYT1 displays a 2:1 :1 stoichiometry and is the main regulator of extracellular glycine concentrations. The neuronal GLYT2, with higher sodium coupling (3:1 :1), supplies glycine to the pre-synaptic terminal to refill synaptic vesicles. In this work, using structural homology modelling and molecular dynamics simulations of GLYTs, we predict the conservation of the two sodium sites present in the template (leucine transporter from Aquifex aeolicus), and confirm its use by mutagenesis and functional analysis. GLYTs Na1 and Na2 sites show differential cation selectivity, as inferred from the action of lithium, a non-transport-supporting ion, on Na(+)-site mutants. GLYTs lithium responses were unchanged in Na1-site mutants, but abolished or inverted in mutants of Na2 site, which binds lithium in the presence of low sodium concentrations and therefore, controls lithium responses. Here, we report, for the first time, that lithium exerts opposite actions on GLYTs isoforms. Glycine transport by GLYT1 is inhibited by lithium whereas GLYT2 transport is stimulated, and this effect is more evident at increased glycine concentrations. In contrast to GLYT1, high and low affinity lithium-binding processes were detected in GLYT2.     

Expression patterns of glycine transporters (xGlyT1, xGlyT2, and xVIAAT) in Xenopus laevis during early development

Glycine, a major inhibitory neurotransmitter in the vertebrate nervous system, not only functions in synaptic signaling, but has also been implicated in regulating neuronal differentiation, neuronal proliferation, synaptic modeling, and neural network stability. Elements of the glycinergic phenotype include the membrane-bound glycine transporters (GLYT1 and GLYT2), which remove glycine from the synaptic cleft, and the vesicular inhibitory amino acid transporter (VIAAT or VGAT), which sequesters both glycine and GABA into synaptic vesicles. Here, we describe the spatial and temporal expression patterns of xGlyT1, xGlyT2, and xVIAAT during early developmental stages of Xenopus laevis. In situ hybridization reveals that xGlyT1 is first expressed in early tailbud stages in the midbrain, hindbrain, and anterior spinal cord; it extends posteriorly through the spinal cord and appears in the forebrain, retina, between the somites, and in the blood islands by swimming tadpole stages. xGlyT2 and xVIAAT initially appear in late neurula stages in the anterior spinal cord. By swimming tadpole stages, the expression of these genes appears in the forebrain, midbrain, and hindbrain and extends posteriorly through the spinal cord; xVIAAT is also expressed in the retina. Confocal analysis of multiplex fluorescent in situ hybridization signal in the spinal cord reveals that xGlyT1 and xGlyT2 share little cellular colocalization. While there is significant coexpression between xVIAAT and xGlyT2, xVIAAT and the GABAergic marker glutamic acid decarboxylase (xGAD67), and xGlyT2 and xGAD67, each gene also appears to have discrete, non-colocalized areas of expression.     

Characteristics and Regulation of Glycine Transport in Bergmann Glia

In the vertebrate CNS, glycine acts as an inhibitory neurotransmitter and as the obligatory coagonist of glutamate at N-methyl-d-aspartate receptors. These roles depend on extracellular glycine levels, regulated by Na⁺/Cl⁻-dependent transporters GLYT1, present mainly in glial cells, and GLYT2, predominantly neuronal. In Bergmann glia, GLYT1 mediates both, glycine uptake and efflux, which, in turn, influences excitatory neurotransmission at Purkinje cell synapses. The biochemical properties of GLYTs and their regulation by signaling pathways in these cells are largely unknown. We characterized Gly uptake in confluent primary cultures of Bergmann glia from chick cerebellum. Transport was found to be energy- and Na⁺-dependent, and was resolved into a high (Km=25 μM) and a low affinity (Km=1.1 mM) components identified as GLYT1 and transport System A, respectively. Results show that high affinity transport by GLYT1 is regulated by calcium from intracellular stores, calmodulin, and myosin light chain kinase through an actin cytoskeleton-mediated action. Special issue dedicated to Dr. Simo S. Oja     

P2Y purinergic regulation of the glycine neurotransmitter transporters

The sodium- and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5'-diphosphate in rat brainstem/spinal cord primary neuronal cultures and adult rat synaptosomes leads to the inhibition of GLYT2 and the stimulation of GLYT1 by a paracrine regulation. These effects are mainly mediated by the ADP-preferring subtypes P2Y(1) and P2Y(13) because the effects are partially reversed by the specific antagonists N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate and pyridoxal-5'-phosphate-6-azo(2-chloro-5-nitrophenyl)-2,4-disulfonate and are totally blocked by suramin. P2Y(12) receptor is additionally involved in GLYT1 stimulation. Using pharmacological approaches and siRNA-mediated protein knockdown methodology, we elucidate the molecular mechanisms of GLYT regulation. Modulation takes place through a signaling cascade involving phospholipase C activation, inositol 1,4,5-trisphosphate production, intracellular Ca(2+) mobilization, protein kinase C stimulation, nitric oxide formation, cyclic guanosine monophosphate production, and protein kinase G-I (PKG-I) activation. GLYT1 and GLYT2 are differentially sensitive to NO/cGMP/PKG-I both in brain-derived preparations and in heterologous systems expressing the recombinant transporters and P2Y(1) receptor. Sensitivity to 2-methylthioadenosine 5'-diphosphate by GLYT1 and GLYT2 was abolished by small interfering RNA (siRNA)-mediated knockdown of nitric-oxide synthase. Our data may help define the role of GLYTs in nociception and pain sensitization.     

Zn2+ inhibits glycine transport by glycine transporter subtype 1b

In the central nervous system, glycine is a co-agonist with glutamate at the N-methyl-D-aspartate subtype of glutamate receptors and also an agonist at inhibitory, strychnine-sensitive glycine receptors. The GLYT1 subtypes of glycine transporters (GLYTs) are responsible for regulation of glycine at excitatory synapses, whereas a combination of GLYT1 and GLYT2 subtypes of glycine transporters are used at inhibitory glycinergic synapses. Zn2+ is stored in synaptic vesicles with glutamate in a number of regions of the brain and is believed to play a role in modulation of excitatory neurotransmission. In this study we have investigated the actions of Zn2+ on the glycine transporters, GLYT1b and GLYT2a, expressed in Xenopus laevis oocytes and we demonstrate that Zn2+ is a noncompetitive inhibitor of GLYT1 but has no effect on GLYT2. We have also investigated the molecular basis for these differences and the relationship between the Zn2+ and proton binding sites on GLYT1. Using site-directed mutagenesis, we identified 2 histidine residues, His-243 in the large second extracellular loop (ECL2) and His-410 in the fourth extracellular loop (ECL4), as two coordinates in the Zn2+ binding site of GLYT1b. In addition, our study suggests that the molecular determinants of proton regulation of GLYT1b are localized to the 2 histidine residues (His-410 and His-421) of ECL4. The ability of Zn2+ and protons to regulate the rate of glycine transport by interacting with residues situated in ECL4 of GLYT1b suggests that this region may influence the substrate translocation mechanism.     

The second intracellular loop of the glycine transporter 2 contains crucial residues for glycine transport and phorbol ester-induced regulation

Na+ and Cl(-)-coupled glycine transporters control the availability of glycine neurotransmitter in the synaptic cleft of inhibitory glycinergic pathways. In this report, we have investigated the involvement of the second intracellular loop of the neuronal glycine transporter 2 (GLYT2) on the protein conformational equilibrium and the regulation by 4alpha-phorbol 12 myristate 13-acetate (PMA). By substituting several charged (Lys-415, Lys-418, and Lys-422) and polar (Thr-419 and Ser-420) residues for different amino acids and monitoring plasma membrane expression and kinetic behavior, we found that residue Lys-422 is crucial for glycine transport. The introduction of a negative charge in 422, and to a lower extent in neighboring N-terminal residues, dramatically increases transporter voltage dependence as assessed by response to high potassium depolarizing conditions. In addition, [2-(trimethylammonium)ethyl] methanethiosulfonate accessibility revealed a conformational connection between Lys-422 and the glycine binding/permeation site. Finally, we show that the mutation of positions Thr-419, Ser-420, and mainly Lys-422 to acidic residues abolishes the PMA-induced inhibition of transport activity and the plasma membrane transporter internalization. Our results establish a new structural basis for the action of PMA on GLYT2 and suggest a complex nature of the PMA action on this glycine transporter.     

Dynamics of forward and reverse transport by the glial glycine transporter, glyt1b

Glycine is a coagonist at the N-methyl-D-aspartate receptor. Changes in extracellular glycine concentration may modulate N-methyl-D-aspartate receptor function and excitatory synaptic transmission. The GLYT1 glycine transporter is present in glia surrounding excitatory synapses, and plays a key role in regulating extracellular glycine concentration. We investigated the kinetic and other biophysical properties of GLYT1b, stably expressed in CHO cells, using whole-cell patch-clamp techniques. Application of glycine produced an inward current, which decayed within a few seconds to a steady-state level. When glycine was removed, a transient outward current was observed, consistent with reverse transport of accumulated glycine. The outward current was enhanced by elevating intracellular or lowering extracellular [Na(+)], and was modulated by changes in extracellular [glycine] and time of glycine application. We developed a model of GLYT1b function, which accurately describes the time course of the transporter current under a range of experimental conditions. The model predicts that glial uptake of glycine will decay toward zero during a sustained period of elevated glycine concentration. This property of GLYT1b may permit spillover from glycinergic terminals to nearby excitatory terminals during a prolonged burst of inhibitory activity, and reverse transport may extend the period of elevated glycine concentration beyond the end of the inhibitory burst.     

Substrate-induced conformational changes of extracellular loop 1 in the glycine transporter GLYT2

The neurotransmitter glycine is removed from the synaptic cleft by two Na(+)-and Cl(-)-dependent transporters, the glial (GLYT1) and neuronal (GLYT2) glycine transporters. GLYT2 lacks a conserved cysteine in the first hydrophilic loop (EL1) that is reactive to [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET) in related transporters. A chimeric GLYT2 (GLYT2a-EL1) that contains GLYT1 sequences in this region, including the relevant cysteine, was sensitive to the reagent, and its sensitivity was decreased by co-substrates. We combined cysteine-specific biotinylation to detect transporter-reagent interactions with MTSET inactivation assays and temperature dependence analysis to study the mechanism by which Cl(-), Na(+), and glycine reduce methanethiosulfonate reagent inhibition. We demonstrate a Na(+) protective effect rather than an increased susceptibility to the reagent exerted by Li(+), as reported for the serotonin transporter. The different inhibition, protection, and reactivation properties between GLYT2a-EL1 and serotonin transporter suggest that EL1 is a source of structural heterogeneity involved in the specific effect of lithium on serotonin transport. The protection by Na(+) or Cl(-) on GLYT2a-EL1 was clearly dependent on temperature, suggesting that EL1 is not involved in ion binding but is subjected to ion-induced conformational changes. Na(+) and Cl(-) were required for glycine protection, indicating the necessity of prior ion interaction with the transporter for the binding of glycine. We conclude that EL1 acts as a fluctuating hinge undergoing sequential conformational changes during the transport cycle.     

Endocytosis of the neuronal glycine transporter GLYT2: role of membrane rafts and protein kinase C-dependent ubiquitination

Glycinergic neurotransmission is terminated by sodium- and chloride-dependent plasma membrane transporters. The neuronal glycine transporter 2 (GLYT2) supplies the terminal with substrate to refill synaptic vesicles containing glycine. This crucial process is defective in human hyperekplexia, a condition that can be caused by mutations in GLYT2. Inhibitory glycinergic neurotransmission is modulated by the GLYT2 exocytosis/endocytosis equilibrium, although the mechanisms underlying the turnover of this transporter remain elusive. We studied GLYT2 internalization pathways and the role of ubiquitination and membrane raft association of the transporter in its endocytosis. Using pharmacological tools, dominant-negative mutants and small-interfering RNAs, we show that the clathrin-mediated pathway is the primary mechanism for constitutive and regulated GLYT2 endocytosis in heterologous cells and neurons. We show that GLYT2 is constitutively internalized from cell surface lipid rafts, remaining associated with rafts in subcellular recycling structures. Protein kinase C (PKC) negatively modulates GLYT2 via rapid and dynamic redistribution of GLYT2 from raft to non-raft membrane subdomains and increasing ubiquitinated GLYT2 endocytosis. This biphasic mechanism is a versatile means to modulate GLYT2 behavior and hence, inhibitory glycinergic neurotransmission. These findings may reveal new therapeutic targets to address glycinergic pathologies associated with alterations in GLYT2 trafficking.     

An aspartate residue in the external vestibule of GLYT2 (glycine transporter 2) controls cation access and transport coupling

Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+-Cl--glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes.     

Arachidonic acid and anandamide have opposite modulatory actions at the glycine transporter,GLYT1a

The GLYT1 subtypes of glycine transporter are expressed in glia surrounding excitatory synapses in the mammalian CNS and may regulate synaptic glycine concentrations required for activation of the NMDA subtypes of glutamate receptor. In this report we demonstrate that the rate of glycine transport by GLYT1 is inhibited by arachidonic acid. The cyclo-oxygenase and lipoxygenase inhibitors indomethacin and nordihydroguaiaretic acid, and the protein kinase C inhibitor staurosporine, had no effect on the extent of arachidonic acid inhibition of transport, which suggests that the inhibitory effects of arachidonic acid result from a direct interaction with the transporter. In contrast to arachidonic acid, its amide derivative, anandamide, and the more stable analogue R1-methanandamide stimulate glycine transport. This stimulation is unlikely to be a secondary effect of cannabinoid receptor stimulation because the cannabinoid receptor agonist WIN 55 212-2 had no effect on transport. We suggest that the stimulatory effects of anandamide on GLYT1 are due to a direct interaction with the transporter.     

Subcellular Localization of the Neuronal Glycine Transporter GLYT2 in Brainstem

The neuronal glycine transporter GLYT2 belongs to the neurotransmitter:sodium:symporter (NSS) family and removes glycine from the synaptic cleft, thereby aiding the termination of the glycinergic signal and achieving the reloading of the presynaptic terminal. The task fulfilled by this transporter is fine tuned by regulating both transport activity and intracellular trafficking. Different stimuli such as neuronal activity or protein kinase C (PKC) activation can control GLYT2 surface levels although the intracellular compartments where GLYT2 resides are largely unknown. Here, by biochemical and immunological techniques in combination with electron and confocal microscopy, we have investigated the subcellular distribution of GLYT2 in rat brainstem tissue, and characterized the vesicles that contain the transporter. GLYT2 is shown to be present in small and larger vesicles that contain the synaptic vesicle protein synaptophysin, the recycling endosome small GTPase Rab11, and in the larger vesicle population, the vesicular inhibitory amino acid transporter VIAAT. Rab5A, the GABA transporter GAT1, synaptotagmin2 and synaptobrevin2 (VAMP2) were not present. Coexpression of a Rab11 dominant negative mutant with recombinant GLYT2 impaired transporter trafficking and glycine transport. Dual immunogold labeling of brainstem synaptosomes showed a very close proximity of GLYT2 and Rab11. Therefore, the intracellular GLYT2 resides in a subset of endosomal membranes and may traffic around several compartments, mainly Rab11-positive endosomes.     

Specific transmembrane segments are selectively delayed at the ER translocon during opsin biogenesis

The neuronal glycine transporter GLYT2 controls the availability of the neurotransmitter in glycinergic synapses, and the modulation of its function may influence synaptic transmission. The active transporter is located in membrane rafts and reaches the cell surface through intracellular trafficking. In the present study we prove that GLYT2 constitutively recycles between the cell interior and the plasma membrane by means of a monensin-sensitive trafficking pathway. Also, a regulated trafficking can be triggered by PMA. We demonstrate that PMA inhibits GLYT2 transport by causing net accumulation of the protein in internal compartments through an increase of the internalization rate. In addition, a small increase of plasma membrane delivery and a redistribution of the transporter to non-raft domains is triggered by PMA. A previously identified phorbol-ester-resistant mutant (K422E) displaying an acidic substitution in a regulatory site, exhibits constitutive traffic but, in contrast with the wild-type, fails to show glycine uptake inhibition, membrane raft redistribution and trafficking modulation by PMA. We prove that the action of PMA on GLYT2 involves PKC (protein kinase C)-dependent and -independent pathways, although an important fraction of the effects are PKC-mediated. We show the additional participation of signalling pathways triggered by the small GTPase Rac1 on PMA action. GLYT2 inhibition by PMA and monensin also take place in brainstem primary neurons and synaptosomes, pointing to a GLYT2 trafficking regulation in the central nervous system.     

Calcium- and syntaxin 1-mediated trafficking of the neuronal glycine transporter GLYT2

Previously we demonstrated the existence of a physical and functional interaction between the glycine transporters and the SNARE protein syntaxin 1. In the present report the physiological role of the syntaxin 1-glycine transporter 2 (GLYT2) interaction has been investigated by using a brain-derived preparation. Previous studies, focused on syntaxin 1-transporter interactions using overexpression systems, led to the postulation that syntaxin is somehow implicated in protein trafficking. Since syntaxin 1 is involved in exocytosis of neurotransmitter and also interacts with GLYT2, we stimulated exocytosis in synaptosomes and examined its effect on surface-expression and transport activity of GLYT2. We found that, under conditions that stimulate vesicular glycine release, GLYT2 is rapidly trafficked first toward the plasma membrane and then internalized. When the same experiments were performed with synaptosomes inactivated for syntaxin 1 by a pretreatment with the neurotoxin Bont/C, GLYT2 was unable to reach the plasma membrane but still was able to leave it. These results indicate the existence of a SNARE-mediated regulatory mechanism that controls the surface-expression of GLYT2. Syntaxin 1 is involved in the arrival to the plasma membrane but not in the retrieval. Furthermore, by using immunogold labeling on purified preparations from synaptosomes, we demonstrate that GLYT2 is present in small synaptic-like vesicles. GLYT2-containing vesicles may represent neurotransmitter transporter that is being trafficked. The results of our work suggest a close correlation between exocytosis of neurotransmitter and its reuptake by transporters.     

Glycine transporters: essential regulators of neurotransmission

Glycine has important neurotransmitter functions at inhibitory and excitatory synapses in the vertebrate central nervous system. The effective synaptic concentrations of glycine are regulated by glycine transporters (GlyTs), which mediate its reuptake into nerve terminals and adjacent glial cells. GlyTs are members of the Na(+)/Cl(-)-dependent transporter family, whose activities and subcellular distributions are regulated by phosphorylation and interactions with other proteins. The analysis of GlyT knockout mice has revealed distinct functions of individual GlyT subtypes in synaptic transmission and provided animal models for two hereditary human diseases, glycine encephalopathy and hyperekplexia. Selective GlyT inhibitors could be of therapeutic value in cognitive disorders, schizophrenia and pain.     

Mechanisms of endoplasmic-reticulum export of glycine transporter-1 (GLYT1)

The GLYT1 (glycine transporter-1) regulates both glycinergic and glutamatergic neurotransmission by controlling the reuptake of glycine at synapses. Trafficking to the cell surface of GLYT1 is critical for its function. In the present paper, by using mutational analysis of the GLYT1 C-terminal domain, we identified the evolutionarily conserved motif R(575)L(576)(X(8))D(585) as being necessary for ER (endoplasmic reticulum) export. This is probably due to its capacity to bind Sec24D, a component of the COPII (coatomer coat protein II) complex. This ER export motif was active when introduced into the related GLYT2 transporter but not in the unrelated VSVG (vesicular-stomatitis virus glycoprotein)-GLYT1 protein in which this motif was mutated but was not transported to the plasma membrane, although this effect was rescued by co-expressing these mutants with wild-type GLYT1. This behaviour suggests that GLYT1 might form oligomers along the trafficking pathway. Cross-linking assays performed in rat brain synaptosomes and FRET (fluorescence resonance energy transfer) microscopy in living cells confirmed the existence of GLYT1 oligomers. In summary, we have identified a motif involved in the ER exit of GLYT1 and, in analysing the influence of this motif, we have found evidence that oligomerization is important for the trafficking of GLYT1 to the cell surface. Because this motif is conserved in the NSS (sodium- and chloride-dependent neurotransmitter transporter) family, it is possible that this finding could be extrapolated to other related transporters.