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The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission by controlling the extracellular concentration of synaptic glycine and the supply of neurotransmitter to the presynaptic terminal. Spinal cord glycinergic neurons present in the dorsal horn diminish their activity in pathological pain conditions and behave as gate keepers of the touch-pain circuitry. The pharmacological blockade of GlyT2 reduces the progression of the painful signal to rostral areas of the central nervous system by increasing glycine extracellular levels, so it has analgesic action. O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-l-serine (ALX1393) and N-[[1-(dimethylamino)cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide (ORG25543) are two selective GlyT2 inhibitors with nanomolar affinity for the transporter and analgesic effects in pain animal models, although with deficiencies which preclude further clinical development. In this report, we performed a comparative ligand docking of ALX1393 and ORG25543 on a validated GlyT2 structural model including all ligand sites constructed by homology with the crystallized dopamine transporter from Drosophila melanogaster. Molecular dynamics simulations and energy analysis of the complex and functional analysis of a series of point mutants permitted to determine the structural determinants of ALX1393 and ORG25543 discrimination by GlyT2. The ligands establish simultaneous contacts with residues present in transmembrane domains 1, 3, 6, and 8 and block the transporter in outward-facing conformation and hence inhibit glycine transport. In addition, differential interactions of ALX1393 with the cation bound at Na1 site and ORG25543 with TM10 define the differential sites of the inhibitors and explain some of their individual features. Structural information about the interactions with GlyT2 may provide useful tools for new drug discovery., ‘Ministerio de Economía y Competitividad’, grant number SAF2017-84235-R (AEI/FEDER, EU) to B.L.-C. and by institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO

In contrast to water soluble enzymes which can be purified and studied while in solution, studies of solute carrier (transporter) proteins require both that the protein of interest is situated in a phospholipid membrane and that this membrane forms a closed compartment. An additional challenge to the study of transporter proteins has been that the transport depends on the transmembrane electrochemical gradients. Baruch I. Kanner understood this early on and first developed techniques for studying plasma membrane vesicles. This advanced the field in that the experimenter could control the electrochemical gradients. Kanner, however, did not stop there, but started to solubilize the membranes so that the transporter proteins were taken out of their natural environment. In order to study them, Kanner then had to find a way to reconstitute them (reinsert them into phospholipid membranes). The scope of the present review is both to describe the reconstitution method in full detail as that has never been done, and also to reveal the scientific impact that this method has had. Kanner’s later work is not reviewed here although that also deserves a review because it too has had a huge impact.

The identity of a glycinergic synapse is maintained presynaptically by the activity of a surface glycine transporter, GlyT2, which recaptures glycine back to presynaptic terminals to preserve vesicular glycine content. GlyT2 loss-of-function mutations cause Hyperekplexia, a rare neurological disease in which loss of glycinergic neurotransmission causes generalized stiffness and strong motor alterations. However, the molecular underpinnings controlling GlyT2 activity remain poorly understood. In this work, we identify the Hedgehog pathway as a robust controller of GlyT2 expression and transport activity. Modulating the activation state of the Hedgehog pathway in vitro in rodent primary spinal cord neurons or in vivo in zebrafish embryos induced a selective control in GlyT2 expression, regulating GlyT2 transport activity. Our results indicate that activation of Hedgehog reduces GlyT2 expression by increasing its ubiquitination and degradation. This work describes a new molecular link between the Hedgehog signaling pathway and presynaptic glycine availability., By modulating the activation state of the Hedgehog pathway, de la Rocha-Muñoz et al demonstrate that Hedgehog signaling controls the expression and transport activity of the neuronal glycine transporter GlyT2. This work begins to reveal a potential link between the Hedgehog signaling pathway and presynaptic glycine availability.

Hyperekplexia is a rare sensorimotor syndrome characterized by pathological startle reflex in response to unexpected trivial stimuli for which there is no specific treatment. Neonates suffer from hypertonia and are at high risk of sudden death due to apnea episodes. Mutations in the human SLC6A5 gene encoding the neuronal glycine transporter GlyT2 may disrupt the inhibitory glycinergic neurotransmission and cause a presynaptic form of the disease. The phenotype of missense mutations giving rise to protein misfolding but maintaining residual activity could be rescued by facilitating folding or intracellular trafficking. In this report, we characterized the trafficking properties of two mutants associated with hyperekplexia (A277T and Y707C, rat numbering). Transporter molecules were partially retained in the endoplasmic reticulum showing increased interaction with the endoplasmic reticulum chaperone calnexin. One transporter variant had export difficulties and increased ubiquitination levels, suggestive of enhanced endoplasmic reticulum-associated degradation. However, the two mutant transporters were amenable to correction by calnexin overexpression. Within the search for compounds capable of rescuing mutant phenotypes, we found that the arachidonic acid derivative N-arachidonoyl glycine can rescue the trafficking defects of the two variants in heterologous cells and rat brain cortical neurons. N-arachidonoyl glycine improves the endoplasmic reticulum output by reducing the interaction transporter/calnexin, increasing membrane expression and improving transport activity in a comparable way as the well-established chemical chaperone 4-phenyl-butyrate. This work identifies N-arachidonoyl glycine as a promising compound with potential for hyperekplexia therapy., Spanish ‘Ministerio de Economía y Competitividad’, grant number SAF2017-84235-R (AEI/ FEDER, EU) to B.L.-C. and by institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO

The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission and plays a key role in regulating nociceptive signal progression. The cholinergic system acting through muscarinic acetylcholine receptors (mAChRs) also mediates important regulations of nociceptive transmission being the M2 subtype the most abundantly expressed in the spinal cord. Here we studied the effect of M2 mAChRs stimulation on GlyT2 function co-expressed in a heterologous system with negligible levels of muscarinic receptor activity. We found GlyT2 is down-regulated by carbachol in a calcium-dependent manner. Different components involved in cell calcium homeostasis were analysed to establish a role in the mechanism of GlyT2 inhibition. GlyT2 down-regulation by carbachol was increased by thapsigargin and reduced by internal store depletion, although calcium release from endoplasmic reticulum or mitochondria had a minor role on GlyT2 inhibition. Our results are consistent with a GlyT2 sensitivity to intracellular calcium mobilized by M2 mAChRs in the subcortical area of the plasma membrane. A crucial role of the plasma membrane sodium calcium exchanger NCX is propose, Spanish ‘Ministerio de Economia y Competitividad’, grant number SAF2017-84235-R (AEI/FEDER, EU) to B.L.-C. and by institutional grants from the Fundacion Ramon Areces and Banco de Santander

The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission and plays a key role in regulating nociceptive signal progression. The cholinergic system acting through muscarinic acetylcholine receptors (mAChRs) also mediates important regulations of nociceptive transmission being the M2 subtype the most abundantly expressed in the spinal cord. Here we studied the effect of M2 mAChRs stimulation on GlyT2 function co-expressed in a heterologous system with negligible levels of muscarinic receptor activity. We found GlyT2 is down-regulated by carbachol in a calcium-dependent manner. Different components involved in cell calcium homeostasis were analysed to establish a role in the mechanism of GlyT2 inhibition. GlyT2 down-regulation by carbachol was increased by thapsigargin and reduced by internal store depletion, although calcium release from endoplasmic reticulum or mitochondria had a minor role on GlyT2 inhibition. Our results are consistent with a GlyT2 sensitivity to intracellular calcium mobilized by M2 mAChRs in the subcortical area of the plasma membrane. A crucial role of the plasma membrane sodium calcium exchanger NCX is proposed.

Two series of new compounds containing 1,2,3-triazole moiety were designed as putative GlyT1 inhibitors aiming the discovery of new hits with activity in cognitive disorders. 1,4-Disubstituted α-hydroxy-1,2,3-triazoles were obtained as racemates in moderate to good yields by the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction (click chemistry) as the key step between propargyl alcohols and aryl azides, previously prepared from anilines or boronic acids. Benzo[c]chromene-triazoles were planned to be obtained by palladium-catalyzed C−H activation using [bis(trifluoroacetoxy)iodobenzene] (PhI(TFA)2) of some α-hydroxy-1,2,3-triazoles, since benzo[c]chromenes are also privileged groups with several biological activities, including to the central nervous system. Unexpectedly, 9H-fluorenes-1,2,3-triazoles, instead of benzo[c]chromene-triazoles, were obtained by Friedel-Crafts alkylation reaction. The two series of compounds were tested for inhibition of the glycine transporter (rat GlyT1 isoform) but only the α-hydroxy-1,2,3-triazole 9b was active (half maximal inhibitory concentration (IC50) = 8.0 µM).

Glycinergic inhibitory neurons of the spinal dorsal horn exert critical control over the conduction of nociceptive signals to higher brain areas. The neuronal glycine transporter 2 (GlyT2) is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft and its activity modulates intra and extracellular glycine concentrations. In this report we show that the stimulation of P2X purinergic receptors with βγ-methylene adenosine 5'-triphosphate induces the up-regulation of GlyT2 transport activity by increasing total and plasma membrane expression and reducing transporter ubiquitination. We identified the receptor subtypes involved by combining pharmacological approaches, siRNA-mediated protein knockdown, and dorsal root ganglion cell enrichment in brainstem and spinal cord primary cultures. Up-regulation of GlyT2 required the combined stimulation of homomeric P2X3 and P2X2 receptors or heteromeric P2X2/3 receptors. We measured the spontaneous glycinergic currents, glycine release and GlyT2 uptake concurrently in response to P2X receptor agonists, and showed that the impact of P2X3 receptor activation on glycinergic neurotransmission involves the modulation of GlyT2 expression or activity. The recognized pro-nociceptive action of P2X3 receptors suggests that the fine-tuning of GlyT2 activity may have consequences in nociceptive signal conduction.

Hyperekplexia or startle disease is a dysfunction of inhibitory glycinergic neurotransmission characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. Although rare, this disorder can have serious consequences, including sudden infant death. One of the most frequent causes of hyperekplexia are mutations in the SLC6A5 gene, encoding the neuronal glycine transporter 2 (GlyT2), a key component of inhibitory glycinergic presynapses involved in synaptic glycine recycling though sodium and chloride-dependent co-transport. Most GlyT2 mutations detected so far are recessive, but two dominant missense mutations have been described. The detailed analysis of these mutations has revealed structural cues on the quaternary structure of GlyT2, and opens the possibility that novel selective pharmacochaperones have potential therapeutic effects in hyperekplexia.

The neuronal glycine transporter GlyT2 is an essential regulator of glycinergic neurotransmission that recaptures glycine in presynaptic terminals to facilitate transmitter packaging in synaptic vesicles. Alterations in GlyT2 expression or activity result in lower cytosolic glycine levels, emptying glycinergic synaptic vesicles and impairing neurotransmission. Lack of glycinergic neurotransmission caused by GlyT2 loss-of-function mutations results in Hyperekplexia, a rare neurological disease characterized by generalized stiffness and motor alterations that may cause sudden infant death. Although the importance of GlyT2 in pathology is known, how this transporter is regulated at the molecular level is poorly understood, limiting current therapeutic strategies. Guided by an unbiased screening, we discovered that E3 ubiquitin ligase Ligand of Numb proteins X1/2 (LNX1/2) modulate the ubiquitination status of GlyT2. The N-terminal RING-finger domain of LNX1/2 ubiquitinates a cytoplasmic C-terminal lysine cluster in GlyT2 (K751, K773, K787 and K791), and this process regulates the expression levels and transport activity of GlyT2. The genetic deletion of endogenous LNX2 in spinal cord primary neurons causes an increase in GlyT2 expression and we find that LNX2 is required for PKC-mediated control of GlyT2 transport. This work identifies, to our knowledge, the first E3 ubiquitin-ligases acting on GlyT2, revealing a novel molecular mechanism that controls presynaptic glycine availability. Providing a better understanding of the molecular regulation of GlyT2 may help future investigations into the molecular basis of human disease states caused by dysfunctional glycinergic neurotransmission, such as hyperekplexia and chronic pain., Ministerio de Economía y Competitividad’, grant number SAF2014-58045-R (MINECO/FEDER, UE) and SAF2017-84235-R (AEI/FEDER, UE) to B.L.-C. and C.A., by Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO

The neuronal glycine transporter GlyT2 is an essential regulator of glycinergic neurotransmission that recaptures glycine in presynaptic terminals to facilitate quantal transmitter packaging in synaptic vesicles. Alterations in GlyT2 expression or activity result in lower cytosolic glycine levels, emptying glycinergic synaptic vesicles and impairing neurotransmission. Lack of glycinergic neurotransmission caused by GlyT2 loss-of-function mutations results in Hyperekplexia, a rare neurological disease characterized by generalized stiffness and motor alterations that may result in sudden infant death. Although the importance of GlyT2 in pathology is known, how this transporter is regulated at the molecular level is poorly understood, limiting current therapeutic strategies. Guided by an unbiased screening, we discovered that the E3 ubiquitin ligase Ligand of Numb protein X1 (LNX1) modulates the ubiquitination status of GlyT2. LNX1 ubiquitinates a cytoplasmic C-terminal lysine cluster in GlyT2 (K751, K773, K787 and K791) through its N-terminal RING-finger domain, and this process regulates the expression levels and transport activity of GlyT2 in neurons. These experiments reveal for the first time the identity of an E3 ubiquitin-ligase acting on GlyT2 and identify a novel regulatory mechanism by which neurons regulate GlyT2 expression and activity.

Glycine, besides exerting essential metabolic functions, is an important inhibitory neurotransmitter in caudal areas of the central nervous system and also a positive neuromodulator at excitatory glutamate-mediated synapses. Glial cells provide metabolic support to neurons and modulate synaptic activity. Six transporters belonging to three solute carrier families (SLC6, SLC38, and SLC7) are capable of transporting glycine across the glial plasma membrane. The unique glial glycine-selective transporter GlyT1 (SLC6) is the main regulator of synaptic glycine concentrations, assisted by the neuronal GlyT2. The five additional glycine transporters ATB

Glycine, besides exerting essential metabolic functions, is an important inhibitory neurotransmitter in caudal areas of the central nervous system and also a positive neuromodulator at excitatory glutamate-mediated synapses. Glial cells provide metabolic support to neurons and modulate synaptic activity. Six transporters belonging to three solute carrier families (SLC6, SLC38, and SLC7) are capable of transporting glycine across the glial plasma membrane. The unique glial glycine-selective transporter GlyT1 (SLC6) is the main regulator of synaptic glycine concentrations, assisted by the neuronal GlyT2. The five additional glycine transporters ATB0,+, SNAT1, SNAT2, SNAT5, and LAT2 display broad amino acid specificity and have differential contributions to glial glycine transport. Glial glycine transporters are divergent in sequence but share a similar architecture displaying the 5 + 5 inverted fold originally characterized in the leucine transporter LeuT. The availability of protein crystals solved at high resolution for prokaryotic and, more recently, eukaryotic homologues of this superfamily has advanced significantly our understanding of the mechanism of glycine transport.

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 Ca(2+)-ATPase (PMCA) isoforms 2 and 3, and Na(+)/Ca(2+)-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.

Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na+/Cl−-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [3H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311–Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H+ and Zn2+ dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo., Dirección General de Enseñanza Superior e Investigación Científica (BFU2005-05931/BMC and BIO2005-05786); Ministerio de Ciencia e Innovación (SAF2008-05436); Comunidad Autónoma de Madrid (11/BCB/010, S-SAL-0253/2006); Ministerio de Economia y Competitividad (SAF2011-28674); Centro de Investigación Biomédica en Red de Enfermedades Raras Intramural Project U-751/U-753; Ramón Areces; Medical Research Council (G0601585); Action Medical Research (1966). The group is member of the European Regional Development Fund Grant BFU2007-30688-E/BFI

This work was supported by the Spanish Dirección General de Enseñanza Superior e Investigación Científica [grant numbers BFU2005-05931/BMC and BIO2005-05786], Ministerio de Ciencia e Innovación [grant number SAF2008-05436], Comunidad Autónoma de Madrid [grant numbers 11/BCB/010 and S-SAL-0253/2006], and an institutional grant from the Fundación Ramón Areces. The research group is a member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educación y Ciencia, Spain and the European Regional Development Fund (ERDF) [grant number BFU2007-30688-E/BFI]. A.M. acknowledges the Comunidad Autónoma-de-Madrid for financial support through the AMAROUTO program to the Fundación Severo Ochoa., 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.

J. Neurochem. (2011) 118, 195–204. Abstract 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.

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.

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.

"This research was originally published in Journal of Biological Chemistry. Esther Arribas-González, Jaime de Juan-Sanz, Carmen Aragón, and Beatriz López-Corcuera. Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia. Journal of Biological Chemistry. 2015; 290:2150-2165. © the American Society for Biochemistry and Molecular Biology.", 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. Weshow 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, This work was supported by Spanish “Ministerio de Economía y Competitividad” Grant SAF2011-28674, by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), and by an institutional grant from the “Fundación Ramón Areces.”

The neurotransmitter glycine is removed from the synaptic cleft by two Na(+)-and Cl(-)-dependent transporters: GLYT1 and GLYT2. GLYT1, expressed in glial processes of glycinergic areas and in glia and neurons of glutamatergic pathways that contain N-methyl-d-aspartate (NMDA) receptors, is essential for regulating glycine levels both at glycinergic and NMDA-containing synapses. GLYT2 is the transporter present in glycinergic neurons and provides cytosolic glycine for vesicular release from glycinergic terminals. GLYT1 is selectively inhibited by the sarcosine derivative N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-propyl]sarcosine (NFPS). In the present report, GLYT1-GLYT2 chimeric transporters have been generated and their inhibition by NFPS has been studied. The introduction of GLYT2 transmembrane domains (TMs) 1 or 3, but not 2, on GLYT1 structure reduced the inhibition potency of NFPS and sarcosine. Binding studies and kinetic analysis of NFPS inhibition indicate lower affinity and smaller sensitivity of the chimeras to the compound. Opposite chimeras containing TM1 or TM3 of GLYT1 on GLYT2 structure became sensitive to NFPS. Individual substitution mutants of GLYT2 TM1 residues on GLYT1 and opposite GLYT1 TM1 residues on GLYT2 indicate that the more N-terminal portion of GLYT1 including residue E40 contributes to NFPS specificity. Our results demonstrate that TM1 and TM3, but not TM2, contain residues involved in the specific action of NFPS on GLYT1.

The functions of the high-affinity glycine transporters (GLYTs) in vivo have been revealed recently using gene-deletion studies. Results from studies of homozygous knockout mice have reinforced the idea that GLYTs might be specific clinical targets to modulate inhibitory glycine-mediated neurotransmission. In addition, molecular and behavioural analysis of heterozygous mice has confirmed the therapeutic potential of GLYT1 inhibitors in the treatment of several neurological and psychiatric disorders.

Glycine exerts multiple functions in the central nervous system, as an inhibitory neurotransmitter through activation of specific, Cl--permeable, ligand-gated ionotropic receptors and as an obligatory co-agonist with glutamate on the activation of N-methyl-D-aspartate (NMDA) receptors. In some areas of the central nervous system, glycine seems to be co-released with gamma-aminobutyric acid (GABA), the main inhibitory amino acid neurotransmitter. The synaptic action of glycine ends by active recapture through sodium- and chloride-coupled glycine transporters located in glial and neuronal plasma membranes, whose structure-function relationship is being studied. The trafficking and plasma membrane expressions of these proteins are controlled by regulatory mechanisms. Glycine transporter inhibitors may find application in the treatment of muscle tone defects, epilepsy, schizophrenia, pain and neurodegenerative disorders. This review deals on recent progress on localization, transport mechanisms, structure, regulation and pharmacology of the glycine transporters (GLYTs).

The subcellular localization of glycine transporters one (GLYT1) and two (GLYT2) stably expressed in PC12 cells has been studied. To facilitate visualization, enhanced green fluorescent protein (GFP) was fused to the amino terminus of both glycine transporters. Functional analysis of the GFP-GLYT1 and GFP-GLYT2 stable cell lines demonstrated that they exhibited high affinity for glycine and the characteristic properties of both glycine transporter subtypes. The GFP-coupled transporters were differently distributed throughout the cell. GFP-GLYT1 was mainly localized on the plasma membrane, whereas most of GFP-GLYT2 was present on large dense-core vesicles and endosomes. Both transporters were absent from the synaptic vesicle population in PC12 cells.

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.

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

Glycine 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.

The effects of ethanol on the function of recombinant glycine transporter 1 (GLYT1) and glycine transporter 2 (GLYT2) have been investigated. GLYT1b and GLYT2a isoforms stably expressed in human embryonic kidney 293 (HEK 293) cells showed a differential behaviour in the presence of ethanol; only the GLYT2a isoform was acutely inhibited. The ‘cut-off’ (alcohols with four carbons) displayed by the n-alkanols on GLYT2a indicates that a specific binding site for ethanol exists on GLYT2a or on a GLYT2a-interacting protein. The non-competitive inhibition of GLYT2a indicates an allosteric modulation by ethanol of GLYT2a activity. Chronic treatment with ethanol caused differential adaptive responses on the activity and the membrane expression levels of these transporters. The neuronal GLYT2a isoform decreased in activity and surface expression and the mainly glial GLYT1b isoform slightly increased in function and surface density. These changes may be involved in some of the modifications of glycinergic or glutamatergic neurotransmitter systems produced by ethanol intoxication. British Journal of Pharmacology (2000) 129, 802–810; doi:10.1038/sj.bjp.0703100

We examined the effects of nine different tricyclic antidepressant drugs on the glycine uptake mediated by the glycine transporter 1b (GLYT1b) and glycine transporter 2a (GLYT2a) stably expressed in human embryonic kidney 293 cells. Desipramine, imipramine, clomipramine, nomifensine and mianserin had no effect on the activity of the glycine transporters. Doxepin, amitriptyline and nortriptyline inhibited the two transporter subtypes to a similar extent. Amoxapine displayed a selective inhibition of GLYT2a behaving as a 10 fold more efficient inhibitor of this isoform than of GLYT1b. Kinetic analysis of the initial rates of glycine uptake by GLYT2a as a function of either glycine, chloride or sodium concentration, in the absence and presence of amoxapine indicated that amoxapine behaved as a competitive inhibitor of both glycine and chloride and a mixed-type inhibitor with respect to sodium. A kinetic model was developed which explains adequately these data, and gives information about the order of binding of sodium and chloride ions to GLYT2a. Our results may contribute to the development of the glycine transporter pharmacology. Additionally, the inhibition of the glycine uptake by GLYT2 is suggested to have some role in the sedative and psychomotor side effects of amoxapine. Keywords: Glycine transporters, stable expression, amoxapine, tricyclic antidepressants, human embryonic kidney cells, transport mechanism Introduction Glycine is a major inhibitory neurotransmitter in the spinal cord and the brain stem of vertebrates, where it participates in a variety of motor and sensory functions. In addition, glycine could potentiate the action of glutamate, the main excitatory neurotransmitter in the brain, on postsynaptic N-methyl-D-aspartate (NMDA) receptors. The re-uptake of glycine into presynaptic nerve terminals and surrounding glial processes plays a major role in the maintenance of low synaptic levels of the transmitter (Iversen, 1971; Kanner & Shuldiner, 1987). Glycine transporters are members of the Na+- and Cl−-dependent neurotransmitter transporter gene family (Liu et al., 1992b; Amara & Kuhar, 1993; Shafqat et al., 1993; Malandro & Kilberg, 1996), a group of integral glycoproteins (Nunez & Aragon, 1994; Tate & Blakely, 1994; Olivares et al., 1995) which share a common structure with 12 transmembrane domains (Kanner & Kleinberger-Doron, 1994). Several neurotransmitter uptake systems, including those for glycine, present an unexpected molecular heterogeneity. By now, two glycine transporter genes (GLYT1 and GLYT2) (Liu et al., 1992a, 1993; Smith et al., 1992; Borowsky et al., 1993; Kim et al., 1994; Adams et al., 1995) have been cloned. GLYT1 presents three isoforms (GLYT1a, GLYT1b and GLYT1c) that differ in their amino terminal sequences and are generated both by alternative promoter usage and by alternative splicing (Smith et al., 1992; Liu et al., 1993; Adams et al., 1995; Borowsky & Hoffman, 1998). Recently, a second GLYT2 isoform (GLYT2b) has been isolated, cloned and characterized in our laboratory (Ponce et al.,1998). The GLYT1 variants are pharmacologically distinguishable from the GLYT2 ones by their higher sensitivity to the inhibition by sarcosine (Liu et al., 1993) but no GLYT2-specific inhibitors are available to date. Amoxapine is a tricyclic dibenzoxazepine (an N-aryl piperazine) which acts similarly to several other tricyclic antidepressants. The clinical effectiveness of some tricyclic antidepressants is now thought to be due to their inhibitory effects on the presynaptic monoamine re-uptake systems. In the case of amoxapine, it has been demonstrated to inhibit norepinephrine transport (Edwards et al., 1988). However, most of the tricyclic antidepressants have a considerable variety of undesired side effects, a pervasive property which can be due to interactions with neurotransmitter receptors. In this paper we examined the effects of several tricyclic antidepressant compounds on the activities of the high affinity glycine transporters GLYT1b and GLYT2a stably expressed in human embryonic kidney 293 (HEK 293) cells. We found that among the tricyclic antidepressants tested, amoxapine inhibited, in a specific manner, the GLYT2a isoform of the glycine transporters. Kinetic analysis of the GLYT2a-mediated glycine uptake in the presence or absence of amoxapine was performed to study the mechanism of amoxapine inhibition. A model was presented which satisfactorily explains the experimental data. Our results indicate that the simultaneous binding of amoxapine and either glycine or chloride to the transporter is not possible although ternary complexes between Na+, inhibitor and transporter may exist, therefore suggesting that Na+ may bind glycine transporter before Cl− and glycine.

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. © 2013 de Juan-Sanz et al., Spanish Direccion General de Investigacion Cientıfica y Tecnica (SAF2008-05436; SAF2011-28674); Fondo de Investigaciones Sanitarias (CIBERER); Comunidad Autonoma de Madrid; Fundacion Ramon Areces

The neuronal transporter GlyT2 is a polytopic, 12-transmembrane domain, plasma membrane glycoprotein involved in the removal and recycling of synaptic glycine from inhibitory synapses. Mutations in the human GlyT2 gene (SLC6A5) that cause deficient glycine transport or defective GlyT2 trafficking are the second most common cause of hyperekplexia or startle disease. In this study we examined several aspects of GlyT2 biogenesis that involve the endoplasmic reticulum chaperone calnexin (CNX). CNX binds transiently to an intermediate under-glycosylated transporter precursor and facilitates GlyT2 processing. In cells expressing GlyT2, transporter accumulation and transport activity were attenuated by siRNA-mediated CNX knockdown and enhanced by CNX overexpression. GlyT2 binding to CNX was mediated by glycan and polypeptide-based interactions as revealed by pharmacological approaches and the behavior of GlyT2 N-glycan-deficient mutants. Moreover, transporter folding appeared to be stabilized by N-glycans. Co-expression of CNX and a fully non-glycosylated mutant rescues glycine transport but not mutant surface expression. Hence, CNX discriminates between different conformational states of GlyT2 displaying a lectin-independent chaperone activity. GlyT2 wild-type and mutant transporters were finally degraded in the lysosome. Our findings provide further insight into GlyT2 biogenesis, and a useful framework for the study of newly synthesized GlyT2 transporters bearing hyperekplexia mutations. © 2013 Arribas-González et al., Direccion General de Ensenanza Superior e Investigacion Cientıfica (BFU2005-05931/BMC and BIO2005-05786); Ministerio de Ciencia e Innovacion (SAF2008-05436); Comunidad Autonoma de Madrid (11/BCB/010 and S-SAL-0253/2006); Ministerio de Economia y Competitividad (SAF2011-28674); CIBERER (intramural project U-751/U-753); Fundacion Ramon Areces

The neuronal glycine transporter GlyT2 plays a fundamental role in the glycinergic neurotransmission by recycling the neurotransmitter to the presynaptic terminal. GlyT2 is the main supplier of glycine for vesicle refilling, a process that is absolutely necessary to preserve quantal glycine content in synaptic vesicles. Alterations in GlyT2 activity modify glycinergic neurotransmission and may underlie several neuromuscular disorders, such as hyperekplexia, myoclonus, dystonia, and epilepsy. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans and produce congenital muscular dystonia type 2 (CMD2) in Belgian Blue cattle. GlyT2 function is strictly coupled to the sodium electrochemical gradient actively generated by the Na+/K+-ATPase (NKA). GlyT2 cotransports 3Na++/Cl-/glycine generating large rises ofNa++inside the presynaptic terminal that must be efficiently reduced by theNKA to preserve Na++homeostasis. In this work, we have used high-throughput mass spectrometry to identify proteins interacting with GlyT2 in the CNS. NKA was detected as a putative candidate and through reciprocal coimmunoprecipitations and immunocytochemistry analyses the association between GlyT2 and NKA was confirmed. NKA mainly interacts with the raft-associated active pool of GlyT2, and low and high levels of the specific NKA ligand ouabain modulate the endocytosis and total expression of GlyT2 in neurons. The ouabainmediated downregulation of GlyT2 also occurs in vivo in two different systems: zebrafish embryos and adult rats, indicating that this NKA-mediated regulatory mechanism is evolutionarily conserved and may play a relevant role in the physiological control of inhibitory glycinergic neurotransmission. © 2013 the authors., Dirección General de Investigación Científica y Técnica (SAF2008-05436, SAF2011-28674); Fondo de Investigaciones Sanitarias (Centro de Investigación Biomédica en Red de Enfermedades Raras); Fundación Ramón Areces

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., This work was supported by the Spanish ‘Ministerio de Ciencia e Innovación’ (SAF2008-05436), the Comunidad Autónoma de Madrid (S-SAL-0253/2006) and by an institutional grant from the ‘Fundación Ramón Areces'.

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.

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 P2Y1 and P2Y13 because the effects are partially reversed by the specific antagonists N6-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. P2Y12 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 Ca2+ 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 P2Y1 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., This work was supported in part by the Spanish Dirección General de Enseñanza Superior e Investigación Científica Grants BFU2005-05931/BMC and BIO2005-05786, Ministerio de Ciencia e Innovación Grant SAF2008-05436, Comunidad Autónoma de Madrid Grants 11/BCB/010 and S-SAL-0253/2006, and by an institutional grant from the Fundación Ramón Areces.

Two novel gamma-aminobutyric acid (GABA) transporters, GAT3 and GAT4, were cloned from the mouse neonatal brain cDNA library and expressed in Xenopus oocytes. Sequence analysis indicated they were members of the Na(+)-dependent neurotransmitter transporter family. The GABA uptake activities were measured in cRNA injected Xenopus oocytes. The Km for GABA uptake by GAT3 was 18 microM and by GAT4 was 0.8 microM. GAT3 also transports beta-alanine and taurine with Km of 28 and 540 microM, respectively. Similarly, GAT4 transports beta-alanine with Km of 99 microM and taurine with a Km of 1.4 mM. The newly cloned GABA transporters were compared with two previously cloned GABA transporters, GAT1 and GAT2, in terms of molecular and pharmacological properties. While GAT1 and GAT4 gene expression were neural specific, GAT2 and GAT3 mRNAs were detected in other tissues such as liver and kidney, in which GAT3 mRNA was especially abundant. The expression of GAT3 mRNA in mouse brain is developmentally regulated, and its mRNA is abundant in neonatal brain but not in adult brain. High affinity GABA transporters GAT1 and GAT4 were more sensitive to inhibition by nipecotic acid. Low affinity GABA transporters GAT2 and GAT3 were inhibited most effectively by betaine and beta-alanine, respectively. The differential tissue distribution and distinct pharmacological properties of those four GABA transporters suggest functional specialization in the mechanisms of GABA transmission termination.

A taurine/beta-alanine transporter was cloned from a mouse brain cDNA library by screening with a partial cDNA probe of the glycine transporter at low stringency. The deduced amino acid sequence predicts 590 amino acids with typical characteristics of the sodium-dependent neurotransmitter transporters such as sequence homology and membrane topography. However, the calculated isoelectric point of the taurine/beta-alanine transporter is more acidic (pI = 5.98) than those (pI > 8.0) of other cloned neurotransmitter transporters. Xenopus oocytes injected with cRNA of the cloned transporter expressed uptake activities with Km = 4.5 microM for taurine and Km = 56 microM for beta-alanine. Northern hybridization showed a single transcript of 7.5 kilobases that was highly enriched in kidney and distributed evenly in various parts of the brain. In situ hybridization showed the mRNA of the taurine/beta-alanine transporter to be localized in the corpus callosum, striatum, and anterior commisure. Specific localization of the taurine/beta-alanine transporter in mouse brain suggests a potential function for taurine and beta-alanine as neurotransmitters.

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

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

The subcellular localization of glycine transporters one (GLYT1) and two (GLYT2) stably expressed in PC12 cells has been studied. To facilitate visualization, enhanced green fluorescent protein (GFP) was fused to the amino terminus of both glycine transporters. Functional analysis of the GFP-GLYT1 and GFP-GLYT2 stable cell lines demonstrated that they exhibited high affinity for glycine and the characteristic properties of both glycine transporter subtypes. The GFP-coupled transporters were differently distributed throughout the cell. GFP-GLYT1 was mainly localized on the plasma membrane, whereas most of GFP-GLYT2 was present on large dense-core vesicles and endosomes. Both transporters were absent from the synaptic vesicle population in PC12 cells.

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.

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.

Publisher Summary This chapter describes a biochemical procedure to obtain a highly purified glycine transport activity with GLYT2-like properties from pig brain stem. The main advantage of this procedure is to provide the transporter in its native state, what readily permits the study of its biochemical, structural, and functional properties. Glycine acts as an inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates, mainly in the spinal cord and the brain stem. An additional role of glycine is the potentiation of glutamate excitatory action on postsynaptic N-methyl-D-aspartate (NMDA) receptors. The reuptake of glycine into presynaptic nerve terminals or neighboring glial cells provides one way of clearing the neurotransmitter from the extracellular space and constitutes an efficient mechanism by which its postsynaptic action can be terminated. This process is carried out by a transport system that actively accumulates glycine and that is energized by the electrochemical gradient of sodium. The purification of glycine transporter involves the solubilization of the transporter protein from plasma membrane vesicles followed by three chromatographic steps on phenyl-Sepharose, wheat germ agglutinin-Sepharose and hydroxylapatite columns, and a final sucrose density gradient fractionation.

A nipecotic acid-resistant gamma-aminobutyric acid (GABA) transporter was cloned from a mouse brain cDNA library. The 2.3-kilobase cDNA clone contains an open reading frame of 1842 nucleotides encoding a protein of 614 amino acids. The predicted amino acid sequence indicates it is a member of the gene family of the sodium-dependent neurotransmitter transporters. The new GABA transporter, named GAT2, is highly homologous to the betaine transporter (BGT1) cloned from canine kidney. However, GAT2 expression in the brain distinguished it from BGT1 which was exclusively expressed in the kidney. The transcripts of GAT2 were found in the cerebral cortex, cerebellum, and brainstem as well as in kidney. Expression of GAT2 in Xenopus oocytes revealed a Km of 79 microM for GABA uptake which is about 10-fold higher than that of the high affinity GABA transporter (GAT1). The pharmacology of GAT2 is different from that of GAT1 because of lack of inhibition by guvacine and nipecotic acid and sensitivity to high concentrations of betaine and beta-alanine. GAT2 transports betaine with a Km of about 200 microM, but no significant transport of beta-alanine could be detected. The presence of mRNA encoding GAT2 in parts of the brain suggests it is a neurotransmitter transporter.

Glycine transporter from rat brain stem and spinal cord is inactivated by specific sulfhydryl reagents. Modification of lysine residues also promotes a decrease of the transporter activity but in a lesser extent than that promoted by thiol group reagents. Mercurials showed a more marked inhibitory effect than maleimide derivatives. SH groups display a similar reactivity for p-chloromercuribenzenesulfonate (pCMBS) and mersalyl in synaptosomal membrane vesicles and proteoliposomes reconstituted with the solubilized transporter. However, different reactivity is observed with N-ethylmaleimide (MalNEt), the greatest effect being attained in membrane vesicles. The rate of inactivation by pCMBS and MalNEt is pseudo-first-order showing time- and concentration-dependence. pCMBS and MalNEt decrease the Vmax for glycine transport and to a lesser extent act on the apparent Km. Treatment with dithiothreitol (DTT) of the transporter modified by pCMBS results in a complete restoration of transporter activity indicating that the effect exercised by the reagent is specific for cysteine residues on the protein. It is concluded that SH groups are involved in the glycine transporter function and that these critical residues are mostly located in a relatively hydrophilic environment of the protein.