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.     

Synthesis and characterization of [¹²⁵I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine transporter 1

In this study, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which predominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide (5) showed excellent inhibitory activity of [(3)H]glycine uptake (IC(50)=2.4 nM). Saturation assay in rat cortical membranes revealed that [(125)I]5 had a single high affinity binding site with a K(d) of 1.54 nM and a B(max) of 3.40 pmol/mg protein. In vitro autoradiography demonstrated that [(125)I]5 showed consistent accumulation with GlyT1 expression. The in vitro binding was greatly inhibited by GlyT1 inhibitors but not by other site ligands, which suggested the high specific binding of [(125)I]5 with GlyT1. In the biodistribution and ex vivo autoradiography studies using mice, [(125)I]5 showed high blood-brain barrier permeability (1.68-2.17% dose/g at 15-60 min) and similar regional brain distribution pattern with in vitro results. In addition, pre-treatment of GlyT1 ligands resulted in significant decrease of [(125)I]5 binding in the GlyT1-rich regions. This preliminary study demonstrated that radio-iodinated 5 is a promising SPECT imaging probe for GlyT1.     

A novel, non-substrate-based series of glycine type 1 transporter inhibitors derived from high-throughput screening

The synthesis and structure-activity relationships (SAR) of a series of indane and tetralin inhibitors of the type 1 glycine transporter, derived from a high-throughput screening (HTS) hit, are described. Key modifications that reduced the 5HT1B receptor affinity of the HTS hit and the P450 2D6 inhibition of subsequent analogues are delineated. While these modifications led to potent and selective GlyT1 inhibitors, HERG affinity and human microsomal clearance remain an issue for this series of compounds.     

Transport activities and expression patterns of glycine transporters 1 and 2 in the developing murine brain stem and spinal cord

Glycine serves as a neurotransmitter in spinal cord and brain stem, where it activates inhibitory glycine receptors. In addition, it serves as an essential co-agonist of excitatory N-methyl-d-aspartate receptors. In the central nervous system, extracellular glycine concentrations are regulated by two specific glycine transporters (GlyTs), GlyT1 and GlyT2. Here, we determined the relative transport activities and protein levels of GlyT1 and GlyT2 in membrane preparations from mouse brain stem and spinal cord at different developmental stages. We report that early postnatally (up to postnatal day P5) GlyT1 is the predominant transporter isoform responsible for a major fraction of the GlyT-mediated [(3)H]glycine uptake. At later stages (≥ P10), however, the transport activity and expression of GlyT2 increases, and in membrane fractions from adult mice both GlyTs contribute about equally to glycine uptake. These alterations in the activities and expression profiles of the GlyTs suggest that the contributions of GlyT1 and GlyT2 to the regulation of extracellular glycine concentrations at glycinergic synapses changes during development.     

Pre-synaptic glycine GlyT1 transporter--NMDA receptor interaction: relevance to NMDA autoreceptor activation in the presence of Mg2+ ions

Rat hippocampal glutamatergic terminals possess NMDA autoreceptors whose activation by low micromolar NMDA elicits glutamate exocytosis in the presence of physiological Mg(2+) (1.2 mM), the release of glutamate being significantly reduced when compared to that in Mg(2+)-free condition. Both glutamate and glycine were required to evoke glutamate exocytosis in 1.2 mM Mg(2+), while dizocilpine, cis-4-[phosphomethyl]-piperidine-2-carboxylic acid and 7-Cl-kynurenic acid prevented it, indicating that occupation of both agonist sites is needed for receptor activation. D-serine mimicked glycine but also inhibited the NMDA/glycine-induced release of [(3H]D-aspartate, thus behaving as a partial agonist. The NMDA/glycine-induced release in 1.2 mM Mg(2+) strictly depended on glycine uptake through the glycine transporter type 1 (GlyT1), because the GlyT1 blocker N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine hydrochloride, but not the GlyT2 blocker Org 25534, prevented it. Accordingly, [(3)H]glycine was taken up during superfusion, while lowering the external concentration of Na(+), the monovalent cation co-transported with glycine by GlyT1, abrogated the NMDA-induced effect. Western blot analysis of subsynaptic fractions confirms that GlyT1 and NMDA autoreceptors co-localize at the pre-synaptic level, where GluN3A subunits immunoreactivity was also recovered. It is proposed that GlyT1s coexist with NMDA autoreceptors on rat hippocampal glutamatergic terminals and that glycine taken up by GlyT1 may permit physiological activation of NMDA pre-synaptic autoreceptors.     

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.     

Sustained saturating level of glycine induces changes in NR2B-containing-NMDA receptor localization in the CA1 region of the hippocampus

Post-synaptic actions of glycine are terminated by specialized transporters. There are two genes encoding glycine transporters, GlyT1 and GlyT2. Glycine acts as a co-agonist at N -methyl- d -aspartate glutamatergic receptors (NMDARs). Blockage of GlyT1 enhances NMDAR function by controlling ambient glycine concentrations. Using whole-cell patch-clamp recordings of acute hippocampal slices, we investigated NMDAR kinetics of CA1 pyramidal neurons of mice expressing 50% of GlyT1 (GlyT1+/−). In this study, we report that the glycine modulatory site of the NMDAR at CA1 synapses is saturated in GlyT1+/− but not in wild-type (WT) mice. We also found that the effect of ifenprodil, a highly selective NR2B-containing-NMDAR antagonist, is significantly reduced at CA1 synapses in GlyT1+/− compared to WT mice while immunoblotting experiments do not show significant differences for NR1, NR2A-B-C-D subunits in both types of mice, suggesting alteration in NR2B-containing-NMDAR localization under a state of chronic saturating level of endogenous glycine. Using a pharmacological approach with MK-801 and DL-TBOA, we discriminated synaptic vis-à-vis extra-synaptic NMDARs. We found that NR2B-containing-NMDARs are expressed at a higher level in the extra-synaptic area of CA1 pyramidal neurons from GlyT1+/− compared to WT mice. Our results demonstrate that chronic saturating level of glycine induces significant changes in NMDAR localization and kinetic. Therefore, results from our study should help to gain a better understanding of the role of glycine in pathological conditions.     

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.     

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.     

Differential effect of the benzophenanthridine alkaloids sanguinarine and chelerythrine on glycine transporters

Glycine transporter inhibitors modulate the transmission of pain signals. Since it is well known that extracts from medicinal plants such as Chelidonium majus exhibit analgesic properties, we investigated the effects of alkaloids typically present in this plant on glycine transporters. We found that chelerythrine and sanguinarine selectively inhibit the glycine transporter GlyT1 with comparable potency in the low micromolar range while berberine shows no inhibition at all. At this concentration both alkaloids only minimally affected transport of the closely related glycine transporter GlyT2, suggesting that the effect is not mediated by the inhibitory activity of these alkaloids on the Na(+)/K(+) ATPase. GlyT1 inhibition was time-dependent, noncompetitive and increased with glycine concentration. While chelerythrine inhibition was reversible, the effect of sanguinarine was resistant to wash out. These results suggest that benzophenanthridine alkaloids interact with glycine transporters and at low micromolar range selectively target glycine transporter GlyT1.     

Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia

Hyperekplexia or startle disease is a rare clinical syndrome characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. This neurological disorder can have serious consequences in neonates, provoking brain damage and/or sudden death due to apnea episodes and cardiorespiratory failure. Hyperekplexia is caused by defective inhibitory glycinergic neurotransmission. Mutations in the human SLC6A5 gene encoding the neuronal GlyT2 glycine transporter are responsible for the presynaptic form of the disease. GlyT2 mediates synaptic glycine recycling, which constitutes the main source of releasable transmitter at glycinergic synapses. Although the majority of GlyT2 mutations detected so far are recessive, a dominant negative mutant that affects GlyT2 trafficking does exist. In this study, we explore the properties and structural alterations of the S512R mutation in GlyT2. We analyze its dominant negative effect that retains wild-type GlyT2 in the endoplasmic reticulum (ER), preventing surface expression. We show that the presence of an arginine rather than serine 512 provoked transporter misfolding, enhanced association to the ER-chaperone calnexin, altered association with the coat-protein complex II component Sec24D, and thereby impeded ER exit. The S512R mutant formed oligomers with wild-type GlyT2 causing its retention in the ER. Overexpression of calnexin rescued wild-type GlyT2 from the dominant negative effect of the mutant, increasing the amount of transporter that reached the plasma membrane and dampening the interaction between the wild-type and mutant GlyT2. The ability of chemical chaperones to overcome the dominant negative effect of the disease mutation on the wild-type transporter was demonstrated in heterologous cells and primary neurons.     

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.     

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.     

Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader

The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 x 10(6) wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC(50) and IC(50) values and rank order potency comparable to the 384-well format assays. Calculated Z' factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 +/- 0.21 and 0.53 +/- 0.22, which were slightly higher (Z'(agonist) = 0.55 +/- 0.33) and lower (Z'(antagonist) = 0.70 +/- 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.     

Neuronal and glial glycine transporters have different stoichiometries

A neurotransmitter transporter can potentially mediate uptake or release of substrate, and its stoichiometry is a key factor that controls the driving force and thus the neurotransmitter flux direction. We have used a combination of electrophysiology and radio-tracing techniques to evaluate the stoichiometries of two glycine transporters involved in glycinergic or glutamatergic transmission. We show that GlyT2a, a transporter present in glycinergic boutons, has a stoichiometry of 3 Na+/Cl-/glycine, which predicts effective glycine accumulation in all physiological conditions. GlyT1b, a glial transporter, has a stoichiometry of 2 Na+/Cl-/ glycine, which predicts that glycine can be exported or imported, depending on physiological conditions. GlyT1b may thus modulate glutamatergic synapses by increasing or decreasing the glycine concentration around N-methyl-D-aspartate receptors (NMDARs).     

Characteristics of glycine transport across the inner blood–retinal barrier

Although glycine plays a pivotal role in neurotransmission and neuromodulation in the retina and is present in high concentration in the retina, the source of retinal glycine is still unclear. The purpose of the present study was to investigate glycine transport across the inner blood–retinal barrier (inner BRB). [¹⁴C]Glycine transport at the inner BRB was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) as an in vitro model of the inner BRB and in vivo vascular injection techniques. [¹⁴C]Glycine uptake by TR-iBRB2 cells was Na⁺- and Cl⁻-dependent, and concentration-dependent with Michaelis–Menten constants of 55.4 μM and 8.02 mM, and inhibited by glycine transporter 1 (GlyT1) and system A inhibitors. These uptake studies suggest that GlyT1 and system A are involved in [¹⁴C]glycine uptake by TR-iBRB2 cells. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA 1 and ATA2) mRNA are expressed in TR-iBRB2 cells. An in vivo study suggested that [¹⁴C]glycine is transported from blood to the retina whereas [¹⁴C]α-methylaminoisobutyric acid, a selective substrate for system A, is not. In conclusion, GlyT1 most likely mediates glycine transport at the inner BRB and is expected to play an important role in regulating the glycine concentration in the neural retina.     

3H]-(R)-NPTS,a radioligand for the type 1 glycine transporter

The synthesis of NPTS, 6, a potent inhibitor of the type 1 glycine transporter (GlyT1) is described, as well as preparation of 6 in optically active and tritiated form for use as a radioligand for affinity displacement assay of GlyT1.     

Molecular mechanisms of glycine transporter GlyT2 mutations in startle disease

Startle disease affects newborn children and involves an exaggerated startle response and muscle hypertonia in response to acoustic or tactile stimuli. The primary cause of startle disease is defective inhibitory glycinergic transmission due to mutations in the postsynaptic glycine receptor (GlyR) α1 subunit gene (GLRA1). However, mutations have also been discovered in the genes encoding the GlyRβ subunit (GLRB) and the presynaptic glycine transporter GlyT2 (SLC6A5). GlyT2 mutations have also been detected in Belgian Blue cattle and Irish Wolfhounds, where they have significant economic and animal welfare impacts.     

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.     

Characteristics of glycine transport across the inner blood–retinal barrier

Although glycine plays a pivotal role in neurotransmission and neuromodulation in the retina and is present in high concentration in the retina, the source of retinal glycine is still unclear. The purpose of the present study was to investigate glycine transport across the inner blood–retinal barrier (inner BRB). [¹⁴C]Glycine transport at the inner BRB was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) as an in vitro model of the inner BRB and in vivo vascular injection techniques. [¹⁴C]Glycine uptake by TR-iBRB2 cells was Na⁺- and Cl⁻-dependent, and concentration-dependent with Michaelis–Menten constants of 55.4 μM and 8.02 mM, and inhibited by glycine transporter 1 (GlyT1) and system A inhibitors. These uptake studies suggest that GlyT1 and system A are involved in [¹⁴C]glycine uptake by TR-iBRB2 cells. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA 1 and ATA2) mRNA are expressed in TR-iBRB2 cells. An in vivo study suggested that [¹⁴C]glycine is transported from blood to the retina whereas [¹⁴C]α-methylaminoisobutyric acid, a selective substrate for system A, is not. In conclusion, GlyT1 most likely mediates glycine transport at the inner BRB and is expected to play an important role in regulating the glycine concentration in the neural retina.