Localization of the glycine transporter GLYT1 in glutamatergic synaptic vesicles

We have previously shown the presence of the glycine transporter GLYT1 in glutamatergic terminals of the rat brain. In this study we present immunohistochemical and biochemical evidence indicating that GLYT1 is expressed not only at the plasma membrane of glutamatergic neurons, but also at synaptic vesicles. Confocal microscopy, immunoblots analysis of a highly purified synaptic vesicle fraction and immunoisolation of synaptic vesicles with anti-synaptophysin antibodies strongly suggested the presence of GLYT1 in synaptic vesicles. Moreover, direct observation with the electron microscope of purified vesicles immunoreacted with anti-GLYT1 and colloidal gold demonstrated that about 40% of the small vesicles of the purified vesicle fraction contained GLYT1. Double labeling for GLYT1 and synaptophysin of this vesicular fraction revealed that more of ninety percent of them were synaptic vesicles. Moreover, a significant part of the GLYT1 containing vesicles (86%) also contained the vesicular glutamate transporter vGLUT1, suggesting a functional role of GLYT1 in a subpopulation of glutamatergic vesicles.     

Localization of Glycine Neurotransmitter Transporter (GLYT2) Reveals Correlation with the Distribution of Glycine Receptor

Abstract: We studied by immunocytochemical localization, the glycine neurotransmitter transporter (GLYT2) in mouse brain, using polyclonal antibodies raised against recombinant N-terminus and loop fusion proteins. Western analysis and immunocytochemistry of mouse brain frozen sections revealed caudal-rostral gradient of GLYT2 distribution with massive accumulation in the spinal cord, brainstem, and less in the cerebellum. Immunoreactivity was detected in processes with varicosities but not cell bodies. A correlation was observed between the pattern we obtained and previously reported strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission accompanying the glycine receptor at the classic inhibitory system in the hindbrain.     

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.     

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 transporter 1 associates with cholesterol-rich membrane raft microdomains

Membrane rafts, the highly-ordered, cholesterol-rich microdomains of the plasma membrane play important roles in cellular functions. In this study, GLYT1-CFP and GLYT2-CFP were constructed, followed by investigation of whether the tagged transporters associate with a fluorescence probe that labels membrane rafts (DilC16) by using Fluorescence Resonance Energy Transfer. A close association was observed between DiIC16 and GLYT1-CFP, but not for GLYT2-CFP. The glycine transport ability of GLYT1 is also highly dependent on the integrity of this area. Together, the results suggest that GLYT1 and membrane rafts are co-localized in the membrane, and that this influences the rate of glycine transport.     

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.     

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.     

Effects of repetitive transcranial magnetic stimulation on ER stress-related genes and glutamate, γ-aminobutyric acid and glycine transporter genes in mouse brain

Repetitive transcranial magnetic stimulation (rTMS) is an emerging therapy for the treatment of psychiatric disorders. However, the mechanisms underlying the therapeutic effects of rTMS are still unclear, limiting its optimisation. Lasting effects suggest changes in disease-related genes, so we conducted gene chip and qRT-PCR analyses of genes associated with psychiatric diseases in the mouse brain at various times following 1, 20, 30 or 40 days of rTMS. Many genes were differentially expressed in the rTMS-treated mouse brain compared to sham controls, including genes encoding neurotransmitter transporters (upregulation of EAAT4, GLAST, GLT-1, GAT2, GAT4, GLYT1 and GLYT2), and endoplasmic reticulum (ER)-stress proteins (downregulation of IRE1α, IRE1β, and XBP1, upregulation of ATF6 and GRP78/Bip). Expression changes in many of these genes were also observed 10 days after the last rTMS treatment. In PC12 cells, rTMS upregulated GRP78/Bip mRNA and enhanced resistance against H₂O₂ stress. These results suggest that rTMS differentially modulates multiple genes associated with psychiatric and neurodegenerative disorders. Sustained changes in the expression of these genes may underlie the therapeutic efficacy of chronic rTMS.     

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.     

The neuronal glycine transporter GLYT2 associates with membrane rafts: functional modulation by lipid environment

The neuronal glycine transporter GLYT2 is a plasma membrane protein that removes the neurotransmitter glycine from the synaptic cleft, thereby aiding the pre-synaptic terminal reloading and the termination of the glycinergic signal. Missense mutations in the gene encoding GLYT2 (SLC6A5) cause hyperekplexia in humans. The activity of GLYT2 seems to be highly regulated. In this report, we demonstrate that GLYT2 is associated with membrane rafts in the plasma membrane of brainstem terminals and neurons. The transporter is localized to Triton X-100-insoluble light synaptosomal membranes together with flotillin-1, a marker protein for membrane rafts, in a methyl-β-cyclodextrin (MβCD)-sensitive manner. In brainstem primary neurons, the GLYT2 punctuate pattern visualized by confocal microscopy was modified by cholesterol depletion with MβCD, unlike other non-raft neuronal markers. GLYT2-associated gold particles were observed by electron microscopy on purified rafts from brainstem synaptosomes. Furthermore, either in brainstem terminals and cultured neurons, the pharmacological reduction of the levels of raft components, cholesterol and sphingomyelin, impairs both the association of GLYT2 with membrane rafts and its transport activity. Thus, GLYT2 may require membrane raft location for optimal function, and therefore the lipid environment may constitute a new mechanism to modulate GLYT2.     

The regulation of glycine transporter GLYT1 is mainly mediated by protein kinase Calpha in C6 glioma cells

Glycine has been shown to possess important functions as a bidirectional neurotransmitter. At synaptic clefts, the concentration of glycine is tightly regulated by the uptake of glycine released from nerve terminals into glial cells by the transporter GLYT1. It has been recently demonstrated that protein kinase C (PKC) mediates the downregulation of GLYT1 activity in several cell systems. However, it remains to be elucidated which subtypes of PKC might be important in the regulation of GLYT1 activity. In this study, we attempted to make clear the mechanism of the phorbol 12-myristate 13-acetate (PMA)-suppressed uptake of glycine in C6 glioma cells which have the native expression of GLYT1. In C6 cells, the expression of PKCα, PKCδ, and PKC of the PMA-activated subtypes was detected. The PMA-suppressed action was fully reversed by the removal of both extracellular and intracellular Ca²⁺. Furthermore, the inhibitory effects of PMA or thymeleatoxin (THX), which is a selective activator of conventional PKC (cPKC), were blocked by the downregulation of all PKCs expressed in C6 cells by long-term incubation with THX, or pretreatment with GF109203X or Gö6983, which are broad inhibitors of PKC, or Gö6976, a selective inhibitor of cPKC. On the other hand, treatment of C6 cells with ingenol, a selective activator of novel PKCs, especially PKCδ and PKC, did not affect the transport of glycine. Silencing of PKCδ expression by using RNA interference or pretreatment with the inhibitor peptide for PKC had no effect on the PMA-suppressed uptake of glycine. Together, these results suggest PKCα to be a crucial factor in the regulation of glycine transport in C6 cells.     

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.     

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.     

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.     

Developmental Expression of GABA Transporters GAT1 and GAT4 Suggests Involvement in Brain Maturation

Abstract: cDNA clones representing four pharmacologically distinct GABA transporters (GAT1–GAT4) were previously identified in mouse brain. Two of these, GAT1 and GAT4, were found to be brain specific. We studied GAT1 and GAT4 in the developing rat brain using polyclonal antibodies against recombinant fusion proteins. Patterns of immunoreactivity were very similar in the embryonic and early postnatal stages for both transporters. However, whereas GAT1 immunoreactivity was detected in distinct patterns in gray matter and growing axons, GAT4 immunoreactivity was found in a subset of radial glial cell fascicles. These patterns usually oriented perpendicularly to the axons expressing GAT1. Our results suggest a transient relationship between GAT4-expressing radial glial elements and GAT1-expressing axons. The presence of GAT1 in the cortical marginal zone and the numerous GAT4-positive fascicles observed in the fetal anterior commissure indicate that both transporters may play a role in processes of brain maturation. Because the beginning of expression for both GAT1 and GAT4 correlates with the expression of the α1 subunit of the GABA receptor, the transporters may be connected with the maturation of adult-type GABAergic inhibitory system in the brain.     

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.     

Characterization of the interactions between the glycine transporters GLYT1 and GLYT2 and the SNARE protein syntaxin 1A

In this study we have examined the effect of the SNARE protein syntaxin 1A on the glycine transporters GLYT1 and GLYT2. Our results demonstrate a functional and physical interaction between both glycine transporters and syntaxin 1A. Co-transfection of syntaxin 1A with GLYT1 or GLYT2 in COS cells resulted in approximately 40% inhibition in glycine transport. This inhibition was reversed by the syntaxin 1A-binding protein, Munc18. Furthermore, immunoprecipitation studies showed a physical interaction between syntaxin 1A and both transporters in COS cells and in rat brain tissue. Finally, we conclude that this physical interaction resulted in a partial removal of the glycine transporters from the plasma membrane as demonstrated by biotinylation studies.     

Differential Properties of Two Stably Expressed Brain-Specific Glycine Transporters

Abstract: Clonal cell lines stably expressing the glial glycine transporter 1b (GLYT1b) and the neuronal glycine transporter 2 (GLYT2) from rat brain have been generated and used comparatively to examine their kinetics, ion dependence, and electrical properties. Differential sensitivity of the transporters to sarcosine is clearly exhibited by the clonal cell lines. GLYT2 transports glycine with higher apparent affinity than GLYT1b and is not inhibited by any assayed compound, as deduced by glycine transport assays and electrophysiological recordings. A sigmoidal Na⁺ dependence of the glycine uptake by the stable cell lines is observed, indicating the involvement of more than one Na⁺ in the transport process. A more cooperative behavior for Na⁺ of GLYT2 than GLYT1b is suggested. One Cl⁻ is required for GLYT1b and GLYT2 transport cycles, although GLYT1b shows three times higher affinity for this ion than GLYT2. The number of expressed transporters was sufficient to allow electrophysiological recordings of the uptake current in the two stable cell lines. GLYT2 exhibits more voltage dependence in both its glycine-evoked current and its capacitive currents recorded in the absence of substrate.     

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.