Glycine transporter dimers: evidence for occurrence in the plasma membrane

Different Na(+)/Cl(-)-dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuT(Aa), as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs.     

Why glycine transporters have different stoichiometries

In the brain, neurons and glial cells compete for the uptake of the fast neurotransmitters, glutamate, GABA and glycine, through specific transporters. The relative contributions of glia and neurons to the neurotransmitter uptake depend on the kinetic properties, thermodynamic coupling and density of transporters but also on the intracellular metabolization or sequestration of the neurotransmitter. In the case of glycine, which is both an inhibitory transmitter and a neuromodulator of the excitatory glutamatergic transmission as a co-agonist of N-methyl D-aspartate receptors, the glial (GlyT1b) and neuronal (GlyT2a) transporters differ at least in three aspects: (i) stoichiometries, (ii) reverse uptake capabilities and (iii) pre-steady-state kinetics. A 3 Na(+)/1 Cl(-)/gly stoichiometry was established for GlyT2a on the basis of a 2 charges/glycine flux ratio and changes in the reversal potential of the transporter current as a function of the extracellular glycine, Na(+) and Cl(-) concentrations. Therefore, the driving force available for glycine uphill transport in neurons is about two orders of magnitude larger than for glial cells. In addition, GlyT2a shows a severe limitation for reverse uptake, which suggests an essential role of GlyT2a in maintaining a high intracellular glycine pool, thus facilitating the refilling of synaptic vesicles by the low affinity, low specificity vesicular transporter VGAT/VIAAT. In contrast, the 2 Na(+)/1 Cl(-)/gly stoichiometry and bi-directional transport properties of GlyT1b are appropriate for the control of the extracellular glycine concentration in a submicromolar range that can modulate N-methyl D-aspartate receptors effectively. Finally, analysis of the pre-steady-state kinetics of GlyT1b and GlyT2a revealed that at the resting potential neuronal transporters are preferentially oriented outward, ready to bind glycine, which suggests a kinetic advantage in the uptake contest.     

Calpain-mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2

The glycine transporter 2 (GlyT2) belongs to the family of Na+/CL--dependent plasma membrane transporters and is localized on the presynaptic terminals of glycinergic neurons. GlyT2 differs from other family members by its extended N-terminal cytoplasmic region. We report that activation of a Ca2+-dependent protease, most likely calpain, in spinal cord synaptosomes or cultured spinal cord neurons, results in partial proteolysis of GlyT2. Regions sensitive to calpain cleavage in vivo are located in the N-terminal and, to a lesser extent, C-terminal regions of the transporter protein. Incubation of a GlyT2 N-terminal fusion protein with spinal cord extract in the presence of calcium followed by protein sequence analysis localized the major N-terminal cleavage site after methionine 156, with a second cleavage site being situated after glycine 164. Interestingly, the size of the N-terminally truncated GlyT2 protein (70 kDa) is similar to that of most other transporter family members, and truncated GlyT2 displayed full transport activity upon expression in HEK293 cells. Our data suggest that Ca2+-triggered proteolysis may contribute to the regulation of GlyT2 trafficking and/or function in the neuronal plasma membrane.     

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.     

Generation of a mouse line expressing Cre recombinase in glycinergic interneurons

In caudal regions of the CNS, glycine constitutes the major inhibitory neurotransmitter. Here, we describe a mouse line that expresses Cre recombinase under the control of a BAC transgenic glycine transporter 2 (GlyT2) promoter fragment. Mating of GlyT2‐Cre mice with the Cre reporter mouse lines Rosa26/LacZ and Rosa26/YFP and analysis of double transgenic offsprings revealed strong transgene activity in caudal regions of the central nervous system, i.e., brain stem and spinal cord. Some additional Cre expression was observed in cortical and cerebellar regions. In brain stem and spinal cord, Cre expressing cells were identified as glycinergic interneurons by staining with GlyT2‐ and glycine‐immunoreactive antibodies; here, >80% of the glycine‐immunoreactive cells expressed the Cre reporter protein. These data indicate that GlyT2‐Cre mice are a useful tool for the genetic manipulation of glycinergic interneurons. genesis 48:437–445, 2010. © 2010 Wiley‐Liss, Inc.     

Calpain Sensitive Regions in the N-terminal Cytoplasmic Domains of Glycine Transporters GlyT1A and GlyT1B

Glycine transporters are members of the Na⁺/Cl⁻ dependent transporter gene family and play crucial roles in regulating inhibitory as well as excitatory neurotransmission. In this report we show that calcium elevation in spinal cord synaptosomes decreases the levels of glycine transporter, GlyT1, N-terminal immunoreactivity, and that this decrease can be blocked by calpain inhibitor. Sequencing of GST fusion proteins containing the N-terminal domains of GlyT1A and B splice variants cleaved with rat recombinant calpain identified calpain cleavage sites after glycine 17 in GlyT1B and N-terminally of the first conserved arginine residue in both GlyT1A and GlyT1B. Expression in HEK293 cells revealed that truncation of the N-terminus of GlyT1 results in significant inhibition of glycine uptake. A syntaxin1A GST fusion protein was able to pull-down N-terminally deleted GlyT1, indicating that calpain cleavage does not eliminate syntaxin1A binding. These results suggest that calpain cleavage may regulate the transport activity/turnover of GlyT1 in vivo by cleaving its N-terminal domain.     

PKCβ-dependent phosphorylation of the glycine transporter 1

The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [³²P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23-40%-inhibition on V_max was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.     

PKCβ-dependent phosphorylation of the glycine transporter 1

The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [³²P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23–40%-inhibition on V_max was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.     

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.     

Cellular localization and subcellular distribution of Unc-33-like protein 6, a brain-specific protein of the collapsin response mediator protein family that interacts with the neuronal glycine transporter 2

Unc-33-like protein (Ulip)6, a brain-specific phosphoprotein of the Ulip/collapsin response mediator protein family, was originally identified in our laboratory by yeast two-hybrid screening using the cytoplasmic N-terminal domain of the neuronal glycine transporter, glycine transporter (GlyT) 2, as a bait. Here, the interaction of Ulip6 with the N-terminal domain of GlyT2 was found to be specific for this member of the Ulip/collapsin response mediator protein family and to involve amino acids 135-184 of GlyT2. In pull-down assays and coimmunoprecipitation experiments with rat spinal cord extract, the presence of phosphatase inhibitors significantly enhanced binding of Ulip6 to GlyT2. Subcellular fractionation of spinal cord and retina homogenates at different developmental stages showed Ulip6 immunoreactivity to be associated with light vesicles that were distinct from GlyT2-containing and synaptic vesicles. Immunocytochemistry revealed punctate Ulip6 immunoreactivity in both somatic regions and processes of cultured spinal neurones; no colocalization with GlyT2 or other synaptic marker proteins was found. In retina, which expresses only GlyT1 but not GlyT2, Ulip6 was detected in the inner plexiform layer and along the somata and processes of selected bipolar, amacrine and ganglion cells. Our data support a model in which Ulip6 transiently interacts with GlyT2 in a phosphorylation-dependent manner.     

Gliosis alters expression and uptake of spinal glial amino acid transporters in a mouse neuropathic pain model

Gliosis is strongly implicated in the development and maintenance of persistent pain states following chronic constriction injury of the sciatic nerve. Here we demonstrate that in the dorsal horn of the spinal cord, gliosis is accompanied by changes in glial amino acid transporters examined by immunoblot, immunohistochemistry and RT-PCR. Cytokines, proinflammatory mediators and microglia increase up to postoperative day (pd) 3 before decreasing on pd 7. Then, spinal glial fibrillary acidic protein increases on pd 7, lasting until pd 14 and later. Simultaneously, the expression of glial amino acid transporters for glycine and glutamate (GlyT1 and GLT1) is reduced on pd 7 and pd 14. Consistent with a reduced expression of GlyT1 and GLT1, high performance liquid chromatography reveals a net increase in the concentration of glutamate and glycine on pd 7 and pd 14 in tissue from the lumbar spinal cord of neuropathic mice. In this study we have confirmed that microglial activation precedes astrogliosis. Such a glial cytoskeletal rearrangement correlates with a marked decrease in glycine and glutamate transporters, which might, in turn, be responsible for the increased concentration of these neurotransmitters in the spinal cord. We speculate that these phenomena might contribute, via over-stimulation of NMDA receptors, to the changes in synaptic functioning that are responsible for the maintenance of persistent pain.     

Presynaptic Control of Glycine Transporter 2 (GlyT2) by Physical and Functional Association with Plasma Membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ Exchanger (NCX)

Fast inhibitory glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, glycine is recovered back to the presynaptic terminal by the neuronal glycine transporter 2 (GlyT2) to maintain quantal glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca²⁺-ATPase (PMCA) isoforms 2 and 3, and Na⁺/Ca²⁺-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2–3·NCX complex would help Na⁺/K⁺-ATPase in controlling local Na⁺ increases derived from GlyT2 activity after neurotransmitter release.     

Constitutive Endocytosis and Turnover of the Neuronal Glycine Transporter GlyT2 Is Dependent on Ubiquitination of a C-Terminal Lysine Cluster

Inhibitory glycinergic neurotransmission is terminated by sodium and chloride-dependent plasma membrane glycine transporters (GlyTs). The mainly glial glycine transporter GlyT1 is primarily responsible for the completion of inhibitory neurotransmission and the neuronal glycine transporter GlyT2 mediates the reuptake of the neurotransmitter that is used to refill synaptic vesicles in the terminal, a fundamental role in the physiology and pathology of glycinergic neurotransmission. Indeed, inhibitory glycinergic neurotransmission is modulated by the exocytosis and endocytosis of GlyT2. We previously reported that constitutive and Protein Kinase C (PKC)-regulated endocytosis of GlyT2 is mediated by clathrin and that PKC accelerates GlyT2 endocytosis by increasing its ubiquitination. However, the role of ubiquitination in the constitutive endocytosis and turnover of this protein remains unexplored. Here, we show that ubiquitination of a C-terminus four lysine cluster of GlyT2 is required for constitutive endocytosis, sorting into the slow recycling pathway and turnover of the transporter. Ubiquitination negatively modulates the turnover of GlyT2, such that increased ubiquitination driven by PKC activation accelerates transporter degradation rate shortening its half-life while decreased ubiquitination increases transporter stability. Finally, ubiquitination of GlyT2 in neurons is highly responsive to the free pool of ubiquitin, suggesting that the deubiquitinating enzyme (DUB) ubiquitin C-terminal hydrolase-L1 (UCHL1), as the major regulator of neuronal ubiquitin homeostasis, indirectly modulates the turnover of GlyT2. Our results contribute to the elucidation of the mechanisms underlying the dynamic trafficking of this important neuronal protein which has pathological relevance since mutations in the GlyT2 gene (SLC6A5) are the second most common cause of human hyperekplexia.     

The glial and the neuronal glycine transporters differ in their reactivity to sulfhydryl reagents

The neuronal (GlyT2) and glial (GlyT1) glycine transporters, two members of the Na(+)/Cl(-)-dependent neurotransmitter transporter superfamily, differ by many aspects, such as substrate specificity and Na(+) coupling. We have characterized under voltage clamp their reactivity toward the membrane impermeant sulfhydryl reagent [2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET). In Xenopus oocytes expressing GlyT1b, application of MTSET reduced to the same extent the Na(+)-dependent charge movement, the glycine-evoked current, and the glycine uptake, indicating a complete inactivation of the transporters following cysteine modification. In contrast, this compound had no detectable effect on the glycine uptake and the glycine-evoked current of GlyT2a. The sensitivities to MTSET of the two transporters can be permutated by suppressing a cysteine (C62A) in the first extracellular loop (EL1) of GlyT1b and introducing one at the equivalent position in GlyT2a, either by point mutation (A223C) or by swapping the EL1 sequence (GlyT1b-EL1 and GlyT2a-EL1) resulting in AFQ <--> CYR modification. Inactivation by MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or GlyT1b, suggesting that the arginine in position +2 reduced the cysteine reactivity. Protection assays indicate that EL1 cysteines are less accessible in the presence of all co-transported substrates: Na(+), Cl(-), and glycine. Application of dithioerythritol reverses the inactivation by MTSET of the sensitive transporters. Together, these results indicate that EL1 conformation differs between GlyT1b and GlyT2a and is modified by substrate binding and translocation.     

Uptake of [(3)H]glycine by synaptosomes of channel

It is known that channel catfish erythrocytes can take up glycine by several distinct transport systems. Further, glycine is an inhibitory neurotransmitter in mammalian brain and spinal cord. Consequently, the uptake of [(3)H]glycine by catfish brain was investigated and found to be a saturable process, dependent on the presence of Na(++) and Cl(--) and sensitive to temperature. A kinetic analysis of transport was performed at 22C. This showed that a high-affinity system existed which exhibited a K(m) of 5.1 (+/- 2. 1) microM. Several structural analogues of glycine were capable of inhibiting uptake in a competitive manner. The most effective inhibitor was sarcosine (IC(50) 5 36 microM). Uptake was also able to be inhibited by harmaline, a drug known to interfere with Na(+)-dependent transport processes. It is concluded that glycine transport by channel catfish brain has much in common with transport by mammalian nervous tissue which is carried out by the membrane carriers GLYT1 and GLYT2. On the other hand, synaptosomal transport differs somewhat from glycine transport by channel catfish erythrocytes.     

Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.     

Postnatal Development of Synaptic Glycine Receptors in Normal and Hyperglycinemic Rats

The postnatal development of glycine synaptic receptors has been studied. Strychnine binding to the synaptic membrane fraction is very low at birth, increases thereafter, and reaches adult values at the 15th day in the brain, and at the 30th day in the spinal cord. Throughout postnatal development, there are more glycine receptors in the spinal cord than in the brain. The development of receptors in the spinal cord displays a pattern similar to that reported previously for the glycine reuptake system in spinal cord slices and in the activity of spinal cord glycine synthase. In rats with experimental hyperglycinemia strychnine binding to spinal cord glycine receptors increases much more rapidly, reaching a level 1.5 times the control value by day 10. When the hyperglycinemia was induced after the 10th postnatal day, however, no effect on the glycine receptors was observed. This increased number of receptors could be explained by an effect of glycine on the synaptic stabilisation process. No changes in the KD for strychnine were observed either during postnatal development or in hyperglycinemic rats. The KD remained approximately 10 nM in the spinal cord and 50 nM in the brain. Results are discussed with respect to the ontogeny of glycinergic synapses and the pathogenesis of nonketotic hyperglycinemia.     

Disorder of the inhibitory glycine receptor: inherited myoclonus in Poll Hereford calves

Inherited congenital myoclonus in Poll Hereford calves is characterized by hyperesthesia and myoclonic jerks of the skeletal musculature that occur spontaneously and in response to sensory stimuli. The symptoms of the disorder suggest a failure of spinal inhibition and are similar to those in subconvulsive strychnine poisoning. Strychnine is a high-affinity antagonist of the synaptic actions of glycine. Our recent biochemical studies revealed a specific and marked deficit in [3H]strychnine binding sites in brain stem and spinal cord membranes from myoclonic calves compared with unaffected controls, reflecting a decrease in inhibitory glycine receptors. Glycine is a major inhibitory neurotransmitter in the mammalian central nervous system, and glycinergic transmission is important for the control of both motor and sensory functions in the spinal cord. In other studies, synaptosomes prepared from affected spinal cord showed a significantly increased ability to accumulate [3H]glycine, indicating an increased capacity of the high-affinity neuronal reuptake system for glycine. In contrast, spinal cord glycine concentrations and stimulus-induced release of endogenous glycine, measured in vitro, were unaltered. The major clinical signs of this myoclonic disorder can be explained by the reported deficiency of inhibitory glycine receptors in brain stem and spinal cord, and future research will be directed toward identifying the nature of the genetic alteration responsible for this deficiency. The characteristics of this bovine receptor abnormality are similar to those described for the mutant spastic mouse.     

The proton/amino acid cotransporter PAT2 is expressed in neurons with a different subcellular localization than its paralog PAT1

The new member of the mammalian amino acid/auxin permease family, PAT2, has been cloned recently and represents an electrogenic proton/amino acid symporter. PAT2 and its paralog, PAT1/LYAAT-1, are transporters for small amino acids such as glycine, alanine, and proline. Our immunodetection studies revealed that the PAT2 protein is expressed in spinal cord and brain. It is found in neuronal cell bodies in the anterior horn in spinal cord and in brain stem, cerebellum, hippocampus, hypothalamus, rhinencephalon, cerebral cortex, and olfactory bulb in the brain. PAT2 is expressed in neurons positive for the N-methyl-d-aspartate subtype glutamate receptor subunit NR1. PAT2 is not found in lysosomes, unlike its paralog PAT1, but is present in the endoplasmic reticulum and recycling endosomes in neurons. PAT2 has a high external proton affinity causing half-maximal transport activation already at a pH of 8.3, suggesting that its activity is most likely not altered by physiological pH changes. Transport of amino acids by PAT2 activity is dependent on membrane potential and can occur bidirectionally; membrane depolarization causes net glycine outward currents. Our data suggest that PAT2 contributes to neuronal transport and sequestration of amino acids such as glycine, alanine, and/or proline, whereby the transport direction is dependent on the sum of the driving forces such as substrate concentration, pH gradient, and membrane potential.     

Uptake of Glycine into Synaptic Vesicles Isolated from Rat Spinal Cord

Glycine was taken up by a synaptic vesicle fraction from spinal cord in a Mg-ATP-dependent manner. The accumulation of glycine was inhibited by carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and nigericin, agents known to destroy the proton gradient across the vesicle membrane. Vesicular uptake of glycine was clearly different from synaptosomal uptake, with respect to both the affinity constant and the effect of Na+, ATP, CCCP, and temperature. Oligomycin and strychnine did not inhibit the vesicular uptake, showing that neither mitochondrial H(+)-ATPase nor binding to strychnine-sensitive glycine receptors was involved. It is suggested that the vesicular uptake of glycine is driven by a proton gradient generated by a Mg2(+)-ATPase. A low concentration of Cl- had little effect on the uptake of glycine, whereas the uptake of glutamate in the same experiment was highly stimulated. High concentrations of gamma-amino-n-butyric acid and beta-alanine inhibited vesicular glycine uptake, but glutamate did not. Accumulation of glycine was found to be fourfold higher in a spinal cord synaptic vesicle fraction than in a vesicle fraction from cerebral cortex.