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.     

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.     

Functional expression of release-regulating glycine transporters GLYT1 on GABAergic neurons and GLYT2 on astrocytes in mouse spinal cord

It is widely accepted that glycine transporters of the GLYT1 type are situated on astrocytes whereas GLYT2 are present on glycinergic neuronal terminals where they mediate glycine uptake. We here used purified preparations of mouse spinal cord nerve terminals (synaptosomes) and of astrocyte-derived subcellular particles (gliosomes) to characterize functionally and morphologically the glial versus neuronal distribution of GLYT1 and GLYT2. Both gliosomes and synaptosomes accumulated [3H]GABA through GAT1 transporters and, when exposed to glycine in superfusion conditions, they released the radioactive amino acid not in a receptor-dependent manner, but as a consequence of glycine penetration through selective transporters. The glycine-evoked release of [3H]GABA was exocytotic from synaptosomes but GAT1 carrier-mediated from gliosomes. Based on the sensitivity of the glycine effects to selective GLYT1 and GLYT2 blockers, the two transporters contributed equally to evoke [3H]GABA release from GABAergic synaptosomes; even more surprising, the 'neuronal' GLYT2 contributed more efficiently than the 'glial' GLYT1 to mediate the glycine effect in [3H]GABA releasing gliosomes. These functional results were largely confirmed by confocal microscopy analysis showing co-expression of GAT1 and GLYT2 in GFAP-positive gliosomes and of GAT1 and GLYT1 in MAP2-positive synaptosomes. To conclude, functional GLYT1 are present on neuronal axon terminals and functional GLYT2 are expressed on astrocytes, indicating not complete selectivity of glycine transporters in their glial versus neuronal localization in the spinal cord.     

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.     

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.     

The scaffolding protein PSD-95 interacts with the glycine transporter GLYT1 and impairs its internalization

Recent evidence indicates that the glycine transporter-1 (GLYT1) plays a role in regulation of NMDA receptor function through tight control of glycine concentration in its surrounding medium. Immunohistochemical studies have demonstrated that, as well as being found in glial cells, GLYT1 is also associated with the pre- and postsynaptic aspects of glutamatergic synapses. In this article, we describe the interaction between GLYT1 and PSD-95 in the rat brain, PSD-95 being a scaffolding protein that participates in the organization of glutamatergic synapses. Mutational analysis reveals that the C-terminal sequence of GLYT1 (-SRI) is necessary for the transporter to interact with the PDZ domains I and II of PSD-95. This C-terminal tripeptide motif also seems to be involved in the trafficking of GLYT1 to the membrane, although this process does not involve PDZ proteins. GLYT1 is able to recruit PSD-95 to the plasma membrane, but it does not affect its clustering. However, the interaction stabilizes this transporter at the plasma membrane, blocking its internalization and producing a significant increase in the V(max) of glycine uptake. We hypothesize that PSD-95 might act as a scaffold for GLYT1 and NMDA receptors, allowing GLYT1 to regulate the concentrations of glycine in the micro-environment of NMDA receptors.     

Developmental Expression of the Glycine Transporters GLYT1 and GLYT2 in Mouse Brain

Abstract: Using immunocytochemical localization, the distribution of the glycine transporters GLYT1 and GLYT2 in the developing mouse brain was studied. GLYT1 and GLYT2 immunoreactivity begins during the period of fiber outgrow and synaptogenesis. GLYT2 is first expressed in spinal and spinothalamic white matter and is followed by the expression of synaptophysin. In the postnatal stages, GLYT2 staining in the white matter disappears, and a punctuated pattern in the gray matter emerges. In contrast, in the fetal brain GLYT1 immunoreactivity coincides with gray matter neuropil and processes of radial glia. GLYT1 is distributed over a much wider area of the brain than GLYT2. However, the distribution of these two GLYTs implies that GLYT1 and GLYT2 operate in concert within the area where both are present. At the day 12 embryo stage, GLYT1 antibodies stain the liver, and later they also react with the pancreas and the gastroduodenal junction. No other organs exhibit significant GLYT1 immunoreactivity. We additionally observed the presence of GLYT1 in rat fetal cerebral cortex and hippocampus, which was not detected in fetal mouse brain. Moreover, GLYT1 immunoreactivity was found in the mouse floor plate and the ventral commissure but was not present in the same regions in rats. These findings suggest possible differences in the expression of GLYT1 between these two species.     

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.     

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.     

The role of N-glycosylation in transport to the plasma membrane and sorting of the neuronal glycine transporter GLYT2

Glycine transporter GLYT2 is an axonal glycoprotein involved in the removal of glycine from the synaptic cleft. To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putative N-glycosylation sites on the transport activity, intracellular traffic in COS cells, and asymmetrical distribution of this protein in polarized Madin-Darby canine kidney (MDCK) cells. Transport activity was reduced by 35-40% after enzymatic deglycosylation of the transporter reconstituted into liposomes. Site-directed mutagenesis of the four glycosylation sites (Asn-345, Asn-355, Asn-360, and Asn-366), located in the large extracellular loop of GLYT2, produced an inactive protein that was retained in intracellular compartments when transiently transfected in COS cells or in nonpolarized MDCK cells. When expressed in polarized MDCK cells, wild type GLYT2 localizes in the apical surface as assessed by transport and biotinylation assays. However, a partially unglycosylated mutant (triple mutant) was distributed in a nonpolarized manner in MDCK cells. The apical localization of GLYT2 occurred by a glycolipid rafts independent pathway.     

N-Arachidonyl-glycine inhibits the glycine transporter, GLYT2a

N-arachidonyl-glycine is one of a series of N-arachidonyl-amino acids that are derived from arachidonic acid. N-arachidonyl-glycine is produced in a wide range of tissues with greatest abundance in the spinal cord. Here we report that N-arachidonyl-glycine is a reversible and non-competitive inhibitor of glycine transport by GLYT2a, but has little effect on glycine transport by GLYT1b or gamma-amino butyric acid transport by GAT1. It has previously been reported that the activity of GLYT2a is down-regulated by protein kinase C and therefore we investigated whether the actions of N-arachidonyl-glycine on GLYT2a are mediated by second messenger systems that lead to the activation of protein kinase C. However, the protein kinase C inhibitor, staurosporine, had no effect on the actions of N-arachidonyl-glycine on GLYT2a. Thus, the actions of N-arachidonyl-glycine are likely to be mediated by a direct interaction with the transporter. We have further defined the pharmacophore by investigating the actions of other N-arachidonyl amino acids as well as the closely related compounds arachidonic acid, anandamide and R1-methanandamide. Arachidonic acid, anandamide and R1-methanandamide have no effect on glycine transport, but N-arachidonyl-l-alanine has similar efficacy at GLYT2a to N-arachidonyl-glycine, and N-arachidonyl-gamma-amino butyric acid is less efficacious. These observations define a novel recognition site for the N-arachidonyl amino acids.     

Constitutive and Regulated Endocytosis of the Glycine Transporter GLYT1b Is Controlled by Ubiquitination

The glycine transporter GLYT1 regulates both glycinergic and glutamatergic neurotransmission by controlling the reuptake of glycine at synapses. Trafficking of GLYT1 to and from the cell surface is critical for its function. Activation of PKC down-regulates the activity of GLYT1 through a mechanism that has so far remained uncharacterized. Here we show that GLYT1b undergoes fast constitutive endocytosis that is accelerated by phorbol esters. Both constitutive and regulated endocytosis occur through a dynamin 2- and clathrin-dependent pathway, accumulating in the transporter in transferrin-containing endosomes. A chimera with the extracellular and transmembrane domains of the nerve growth factor receptor and the COOH-terminal tail of GLYT1 was efficiently internalized through this clathrin pathway, suggesting the presence of molecular determinants for GLYT1b endocytosis in its COOH-terminal tail. Extensive site-directed mutagenesis in this region of the chimera highlighted the involvement of lysine residues in its internalization. In the context of the full-length transporter, lysine 619 played a prominent role in both the constitutive and phorbol 12-myristate 13-acetate-induced endocytosis of GLYT1b, suggesting the involvement of ubiquitin modification of GLYT1b during the internalization process. Indeed, we show that GLYT1b undergoes ubiquitination and that this process is stimulated by phorbol 12-myristate 13-acetate. In addition, this endocytosis is impaired in an ubiquitination-deficient cell line, further evidence that constitutive and regulated endocytosis of GLYT1b is ubiquitin-dependent. It remains to be determined whether GLYT1b recycling might be affected in pathologies involving alterations to the ubiquitin system, thereby interfering with its influence on inhibitory and excitatory neurotransmission.Glycine fulfills a dual role in neurotransmission by mediating inhibition through the strychnine-sensitive glycine receptor and excitation as a co-agonist of the NMDA² receptors (1, 2). Although it was initially believed that the concentration of glycine in the synaptic cleft would be sufficient to saturate the glycine sites on NMDA receptors, recent pharmacological and electrophysiological evidence indicates that due to the activity of the GLYT1 (glycine transporter-1) glycine transporter, this is probably not the case. Three isoforms of GLYT1 exist that differ in their NH₂-terminal sequence (GLYT1a, GLYT1b, and GLYT1c), and they are strongly expressed in glycinergic areas of the nervous system, predominantly in glial cells (3). Indeed, mice lacking GLYT1 have impaired glycinergic neurotransmission, which has been attributed to an increase in extracellular glycine close to the strychnine-sensitive glycine receptor (4). Moreover, GLYT1 has been identified in neuronal elements closely associated with the glutamatergic pathways throughout the brain (5, 6). GLYT1 is enriched in presynaptic buttons, where it largely co-localizes with the vesicular glutamate transporter vGLUT1. It is also present in the postsynaptic densities of asymmetric synapses, and complexes containing both NMDA receptor and GLYT1 have been shown to exist (5). In these postsynaptic sites, the distribution of GLYT1 is partially controlled through its interaction with the scaffolding protein PSD-95 (7). Accordingly, GLYT1 is believed to play a role in controlling the concentration of glycine in the microenvironment around the NMDA receptor. Indeed, functional studies have shown that a specific GLYT1 inhibitor, N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine, potentiates NMDA-mediated responses in vitro and in vivo (8–10). The potential role of GLYT1 in glutamatergic neurotransmission has also been confirmed in heterozygous Glyt1^+/− animals that express only 50% of the normal levels of GLYT1 as well as when GLYT1 expression is disturbed in forebrain neurons. In these animals, hippocampal NMDA receptor function is enhanced, and the mice appear to display better memory retention than wild type mice (11–13).The mechanisms responsible for the insertion of GLYT1 into glutamatergic synapses are unknown. However, recent studies indicate that the movement of transporters within the cell is highly organized and that a number of ancillary proteins control their intracellular trafficking by interacting with targeting motifs in the transporter. Indeed, like other neurotransmitter transporters, GLYT1 is asymmetrically distributed in polarized cells (14, 15). The asymmetric distribution of sodium-dependent neurotransmitter transporters (NSS) requires a number of steps that commence with their efficient exit from the endoplasmic reticulum. This is followed by sorting processes in the Golgi complex, insertion into the plasma membrane, and the retention of the transporter at functional synaptic sites. Moreover, the amount of transporter in the plasma membrane is also regulated by endocytosis and recycling mechanisms. Like several other members of the NSS family, GLYT1 is subjected to regulation by protein kinase C. Activation of PKC by phorbol esters down-regulates GLYT1, which is endocytosed from the plasma membrane to intracellular compartments in several cell lines (16–18). For years, the molecular mechanisms that mediate phorbol 12-myristate 13-acetate (PMA)-stimulated endocytosis of the NSS family members have remained elusive. However, recent evidence obtained for the dopamine transporter (DAT) has revealed the importance of ubiquitination of DAT for its endocytosis (19). DAT is ubiquitinated by the Nedd4-2 ligase at several intracellular lysines. Indeed, mutation of these lysines abolished both ubiquitination and phorbol ester-stimulated endocytosis, indicating that the associated ubiquitin molecules serve as a platform to recruit endocytotic adaptors (20–23). Ubiquitination has also been implicated in the endocytosis of other membrane proteins, including the main transporter for glutamate, GLT1, and the system A transporter SNAT2 (24–26).Ubiquitin coupling can involve either mono- or polyubiquitination. Monoubiquitination occurs when a single ubiquitin molecule is coupled to one or more lysine residues on a target protein, such that the final stoichiometry is one ubiquitin per lysine. Polyubiquitination refers to the coupling of a chain of ubiquitins to a lysine on the target protein, with a final stoichiometry of four or more ubiquitins per lysine. Whereas monoubiquitinated proteins are degraded in lysosomes, polyubiquitinated proteins are recognized by and subsequently degraded by the 26 S proteasome (27).In this study, we show that GLYT1b is endocytosed through a clathrin-dependent mechanism, a process that is accelerated by phorbol esters. Through a mutational analysis, we have identified a lysine residue in the COOH-terminal tail of the protein as the major determinant for GLYT1b internalization through both constitutive and PMA-stimulated pathways. Ubiquitination GLYT1b is stimulated by PMA, a finding compatible with ubiquitin being the platform on which the clathrin network is assembled.     

Extracellular Loops 2 and 4 of GLYT2 Are Required for N-Arachidonylglycine Inhibition of Glycine Transport

Concentrations of extracellular glycine in the central nervous system are regulated by Na⁺/Cl⁻-dependent glycine transporters, GLYT1 and GLYT2. N-Arachidonylglycine (NAGly) is an endogenous inhibitor of GLYT2 with little or no effect on GLYT1 and is analgesic in rat models of neuropathic and inflammatory pain. Understanding the molecular basis of NAGly interactions with GLYT2 may allow for the development of novel therapeutics. In this study, chimeric transporters were used to determine the structural basis for differences in NAGly sensitivity between GLYT1 and GLYT2 and also the actions of a series of related N-arachidonyl amino acids. Extracellular loops 2 and 4 of GLYT2 are important in the selective inhibition of GLYT2 by NAGly and by the related compounds N-arachidonyl-γ-aminobutyric acid and N-arachidonyl-d-alanine, whereas only the extracellular loop 4 of GLYT2 is required for N-arachidonyl-l-alanine inhibition of transport. These observations suggest that the structure of the head group of these compounds is important in determining how they interact with extracellular loops 2 and 4 of GLYT2. Site-directed mutagenesis of GLYT2 EL4 residues was used to identify the key residues Arg⁵³¹, Lys⁵³², and Ile⁵⁴⁵ that contribute to the differences in NAGly sensitivity.     

Modulation of a Recombinant Glycine Transporter (GLYT1b) by Activation of Protein Kinase C

Abstract: Treatment of human embryonic kidney cells (HEK 293 cells) expressing the mouse glycine transporter 1 (GLYT1b) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) decreased specific [³H]glycine uptake. This down-regulation resulted from a reduction of the maximal transport rate and was blocked by the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine. The inhibitory effect of PMA treatment was also observed after removing all five predicted phosphorylation sites for PKC in GLYT1b by site-directed mutagenesis. These data indicate that glycine transport by GLYT1b is modulated by PKC activation; however, this regulation may involve indirect phosphorylation mechanisms.     

Glycine is taken up through GLYT1 and GLYT2 transporters into mouse spinal cord axon terminals and causes vesicular and carrier-mediated release of its proposed co-transmitter GABA

Glycine and GABA are likely co-transmitters in the spinal cord. Their possible interactions in presynaptic terminals have, however, not been investigated. We studied the effects of glycine on GABA release using superfused mouse spinal cord synaptosomes. Glycine concentration dependently elicited [(3)H]GABA release which was insensitive to strychnine or 5,7-dichlorokynurenic acid, but was Na(+) dependent and sensitive to the glycine uptake blocker glycyldodecylamide. The glycine effect was external Ca(2+) independent, but was reduced when intraterminal Ca(2+) was chelated with 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid or depleted with thapsigargin, or when vesicular storage was impaired with bafilomycin. Glycine-induced [(3)H]GABA release was prevented, in part, by blocking GABA transport. The glycine effect was halved by sarcosine, a GLYT1 substrate/inhibitor, or by amoxapine, a GLYT2 blocker, and abolished by a mixture of the two. The sensitivity to sarcosine, used as a transporter inhibitor or substrate, persisted in synaptosomes prelabelled with [(3)H]GABA in the presence of beta-alanine, excluding major gliasome involvement. To conclude, in mice spinal cord, transporters for glycine (both GLYT1 and GLYT2) and for GABA coexist on the same axon terminals. Activation of the glycine transporters elicits GABA release, partly by internal Ca(2+)-dependent exocytosis and partly by transporter reversal.     

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.     

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.     

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.     

Mutations within the human GLYT2 (SLC6A5) gene associated with hyperekplexia

Hereditary hyperekplexia is a neuromotor disorder characterized by exaggerated startle reflexes and muscle stiffness in the neonate. The disease has been associated with mutations in the glycine receptor subunit genes GLRA1 and GLRB. Here, we describe mutations within the neuronal glycine transporter 2 gene (GLYT2, or SLC6A5, ) of hyperekplexia patients, whose symptoms cannot be attributed to glycine receptor mutations. One of the GLYT2 mutations identified causes truncation of the transporter protein and a complete loss of transport function. Our results are consistent with GLYT2 being a disease gene in human hyperekplexia.     

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.