Role of palmitoylation on the neuronal glycine transporter GlyT2.

The neuronal glycine transporter GlyT2 removes glycine from the synaptic cleft through active Na+, Cl-, and glycine cotransport contributing to the termination of the glycinergic signal as well as supplying substrate to the presynaptic terminal for the maintenance of the neurotransmitter content in synaptic vesicles. Patients with mutations in the human GlyT2 gene (SLC6A5), develop hyperekplexia or startle disease (OMIM 149400), characterized by hypertonia and exaggerated startle responses to trivial stimuli that may have lethal consequences in the neonates as a result of apnea episodes. Post-translational modifications in cysteine residues of GlyT2 are an aspect of structural interest we analyzed. Our study is compatible with a reversible and short-lived S-acylation in spinal cord membranes, detectable by biochemical and proteomics methods (acyl-Rac binding and IP-ABE) confirmed with positive and negative controls (palmitoylated and non-palmitoylated proteins). According to a short-lived modification, direct labeling using click chemistry was faint but mostly consistent. We have analyzed the physiological properties of a GlyT2 mutant lacking the cysteines with high prediction of palmitoylation and the mutant is less prone to be included in lipid rafts, an effect also observed upon treatment with the palmitoylation inhibitor 2-bromopalmitate. This work demonstrates there are determinants of lipid raft inclusion associated with the GlyT2 mutated cysteines, which are presumably modified by palmitoylation.

E3 ubiquitin ligases LNX1 and LNX2 are major regulators of the presynaptic glycine transporter GlyT2.

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.

[Molecular bases of hereditary hyperekplexia]. / Bases moleculares de la hiperplexia hereditaria.

INTRODUCTION: Hereditary hyperekplexia is a rare clinical syndrome typically characterized by sudden and generalized startle in response to trivial but unexpected tactile or acoustic stimulations. Typically it is accompanied by a temporally but complete muscular rigidly, and usually it manifests shortly after birth. Some affected infants die suddenly from lapses in cardiorespiratory function. Mental development usually is normal. AIM: To summarize and update the molecular bases underlying the hereditary hyperekplexia syndrome. DEVELOPMENT: Approximately 30% of the individuals suffering hereditary hyperekplexia show mutations on a gene located on chromosome 5q32 with a dominant or recessive trait. This gene encodes the alpha subunit of the strychnine-sensitive glycine receptor, which plays a crucial role in inhibitory glycinergic neurotransmission that process sensory and motor information. About 70% of the patients with hyperekplexia do not show genetic defects in the glycine receptor gene; this suggested that additional genes might be affected in this disease. Recent studies have reveals that mutations in the neuronal glycine transporter GLYT2 are a second major cause of hyperekplexia. CONCLUSIONS: Hereditary hyperekplexia is a complex genetic disease in which several genes can be implicated, all of them directly or indirectly involved in inhibitory glycinergic neurotransmission. Two major proteins involved in hyperekplexia are the strychnine-sensitive glycine receptor (GlyR) and the neuronal glycine transporter GLYT2. Implication of secondary additional accompanying or interacting proteins in glycinergic terminals are not ruled out.

Bases moleculares de la hiperplexia hereditaria / Molecular bases of hereditary hyperekplexia

Introducción. La hiperplexia es un síndrome clínico poco común caracterizado por sobresaltos enérgicos y generalizados en respuesta a estímulos triviales generalmente acústicos o táctiles. La hiperplexia hereditaria se manifiesta justo despuésdel nacimiento y los afectados tienen, durante el período perinatal, un alto riesgo de sufrir muerte súbita, debido a episodios de espasmos laríngeos y fallos cardiorrespiratorios. Objetivo. Revisar las bases genéticas y moleculares, conocidas hasta el momento, subyacentes a la hiperplexia hereditaria. Desarrollo. Aproximadamente en el 30% de los pacientes con hiperplexia hereditaria se ha identificado una mutación génica en el cromosoma 5q32. Este gen codifica la subunidad alfa del receptor de glicina sensible a estricnina, el cual regula el tono muscular en el tallo cerebral y la médula espinal, zonas donde desempeñan un papel fundamental las interneuronas inhibidoras glicinérgicas. El 70% de los pacientes con hiperplexia hereditaria no tiene mutaciones en el receptor de glicina, lo que sugiere que otros genes podrían estar implicados en la enfermedad. Recientemente se han encontrado mutaciones en el gen humano del transportador neuronal de glicina GLYT2 en familias con miembros diagnosticados de hiperplexia. Conclusiones. La hiperplexia hereditaria es una enfermedad genética compleja, en la que intervienen diferentes genes que codifican proteínas de vías glicinérgicas inhibidoras. Las proteínas más importantes implicadas en la hiperplexia son el receptor de glicina (GlyR) y el transportador neuronal de glicina GLYT2. No sedescarta la implicación en la enfermedad de otras proteínas que interaccionen con GlyR o con GLYT2, tales como proteínas asociadas, proteínas de andamiaje o proteínas reguladoras

Introduction. Hereditary hyperekplexia is a rare clinical syndrome typically characterized by sudden and generalized startle in response to trivial but unexpected tactile or acoustic stimulations. Typically it is accompanied by a temporally but complete muscular rigidly, and usually it manifests shortly after birth. Some affected infants die suddenly from lapses incardiorespiratory function. Mental development usually is normal. Aim. To summarize and update the molecular bases underlying the hereditary hyperekplexia syndrome. Development. Approximately 30% of the individuals suffering hereditary hyperekplexia show mutations on a gene located on chromosome 5q32 with a dominant or recessive trait. This gene encodes the alpha subunit of the strychnine-sensitive glycine receptor, which plays a crucial role in inhibitory glycinergic neurotransmissionthat process sensory and motor information. About 70% of the patients with hyperekplexia do not show geneticdefects in the glycine receptor gene; this suggested that additional genes might be affected in this disease. Recent studies have reveals that mutations in the neuronal glycine transporter GLYT2 are a second major cause of hyperekplexia. Conclusions. Hereditary hyperekplexia is a complex genetic disease in which several genes can be implicated, all of them directly orindirectly involved in inhibitory glycinergic neurotransmission. Two major proteins involved in hyperekplexia are the strychninesensitive glycine receptor (GlyR) and the neuronal glycine transporter GLYT2. Implication of secondary additional accompanyingor interacting proteins in glycinergic terminals are not ruled out

Regulation of glycine transporters.

The regulation of neurotransmitter transporters is a central aspect of their physiology. Recent studies that focused on syntaxin-1 transporter interactions led to the postulation that syntaxin-1 is somehow implicated in protein trafficking. Because syntaxin-1 is involved in the exocytosis of neurotransmitters and it interacts with glycine transporter 2 (GLYT2), we stimulated exocytosis in synaptosomes and examined its effect on GLYT2 surface-expression and transport activity. We found that GLYT2 is rapidly trafficked first towards the plasma membrane and then internalized under conditions that stimulate vesicular glycine release. However, when syntaxin-1 was inactivated by pre-treatment of synaptosomes with the botulinum neurotoxin C, GLYT2 was unable to reach the plasma membrane but still was able to leave it. These results indicate the existence of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated regulatory mechanism that controls the surface expression of GLYT2. Syntaxin-1 is involved in the transport of GLYT2 to, but not its retrieval from, the plasma membrane. Immunogold-labelling on purified vesicular preparations from synaptosomes showed that GLYT2 is present in small synaptic-like vesicles. This may represent neurotransmitter transporter that is being trafficked. The subcellular distribution of the glycine transporters was further examined in PC12 cells that were stably transfected with the fusions of GLYT1 and GLYT2 with green fluorescent protein. There was a clear difference in their intracellular distribution, GLYT1 being present mainly on the plasma membrane and GLYT2 being localized mainly on large, dense-core vesicles. We are trying to find signal sequences responsible for this differential localization.

Substrate-induced conformational changes of extracellular loop 1 in the glycine transporter GLYT2.

The neurotransmitter glycine is removed from the synaptic cleft by two Na(+)-and Cl(-)-dependent transporters, the glial (GLYT1) and neuronal (GLYT2) glycine transporters. GLYT2 lacks a conserved cysteine in the first hydrophilic loop (EL1) that is reactive to [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET) in related transporters. A chimeric GLYT2 (GLYT2a-EL1) that contains GLYT1 sequences in this region, including the relevant cysteine, was sensitive to the reagent, and its sensitivity was decreased by co-substrates. We combined cysteine-specific biotinylation to detect transporter-reagent interactions with MTSET inactivation assays and temperature dependence analysis to study the mechanism by which Cl(-), Na(+), and glycine reduce methanethiosulfonate reagent inhibition. We demonstrate a Na(+) protective effect rather than an increased susceptibility to the reagent exerted by Li(+), as reported for the serotonin transporter. The different inhibition, protection, and reactivation properties between GLYT2a-EL1 and serotonin transporter suggest that EL1 is a source of structural heterogeneity involved in the specific effect of lithium on serotonin transport. The protection by Na(+) or Cl(-) on GLYT2a-EL1 was clearly dependent on temperature, suggesting that EL1 is not involved in ion binding but is subjected to ion-induced conformational changes. Na(+) and Cl(-) were required for glycine protection, indicating the necessity of prior ion interaction with the transporter for the binding of glycine. We conclude that EL1 acts as a fluctuating hinge undergoing sequential conformational changes during the transport cycle.

Glycine neurotransmitter transporters: an update.

Glycine accomplishes several functions as a transmitter in the central nervous system (CNS). As an inhibitory neurotransmitter, it participates in the processing of motor and sensory information that permits movement, vision, and audition. This action of glycine is mediated by the strychnine-sensitive glycine receptor, whose activation produces inhibitory post-synaptic potentials. In some areas of the CNS, glycine seems to be co-released with GABA, the main inhibitory amino acid neurotransmitter. In addition, glycine modulates excitatory neurotransmission by potentiating the action of glutamate at N-methyl-D-aspartate (NMDA) receptors. It is believed that the termination of the different synaptic actions of glycine is produced by rapid re-uptake through two sodium-and-chloride-coupled transporters, GLYT1 and GLYT2, located in the plasma membrane of glial cells or pre-synaptic terminals, respectively. Glycine transporters may become major targets for therapeutic of pathological alterations in synaptic function. This article reviews recent progress on the study of the molecular heterogeneity, localization, function, structure, regulation and pharmacology of the glycine transporter proteins.

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.

Calcium- and syntaxin 1-mediated trafficking of the neuronal glycine transporter GLYT2.

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

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

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

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

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

Differential effects of ethanol on glycine uptake mediated by the recombinant GLYT1 and GLYT2 glycine transporters.

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.

Differential effects of the tricyclic antidepressant amoxapine on glycine uptake mediated by the recombinant GLYT1 and GLYT2 glycine transporters.

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. British Journal of Pharmacology (2000) 129, 200 - 206

Differential properties of two stably expressed brain-specific glycine transporters.

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.

Cloning and expression of a spinal cord- and brain-specific glycine transporter with novel structural features.

A novel glycine transporter (GLYT2) was cloned from a rat brain cDNA library. GLYT2 is about 48 and 50% homologous to the previously cloned mouse glycine transporter (GLYT1) and rat proline transporter (PROT), respectively. GLYT2 differs from GLYT1 in molecular structure, tissue specificity, and pharmacological properties. The cDNA of GLYT2 encodes for 799 amino acid residues with an extended amino-terminal peptide containing 200 amino acids before the first transmembrane domain. Potential phosphorylation sites for protein kinase C, cAMP-dependent kinase, and calmodulin-dependent kinase were identified in the amino-terminal region. GLYT2 mRNA was shown to be specifically localized in spinal cord, brain stem, and to a lesser extent in the cerebellum. In contrast, GLYT1 mRNA distribution in the brain has been found previously to be more ubiquitous. Xenopus oocytes injected with GLYT2 cRNA transport glycine with a Km of 17 microM, and the uptake of glycine is resistant to inhibition by sarcosine. The experimental data suggests GLYT2 might play a major role in the termination of the inhibitory effect of glycine in the brain stem and spinal cord of vertebrates. On the other hand, the main function of GLYT1 may be in the modulation of excitatory nerve terminals. Two types of GLYT1 cDNA, GLYT1a and GLYT1b, were cloned from the mouse brain library. They differ only at their amino-terminal sequences, and GLYT1b contains two additional potential phosphorylation sites for proline-dependent kinase. Cloning of the gene encoding the GLYT1 revealed that the two variants resulted from a differential splicing.

Molecular characterization of four pharmacologically distinct gamma-aminobutyric acid transporters in mouse brain [corrected].

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.

Hydrodynamic properties and immunological identification of the sodium- and chloride-coupled glycine transporter.

We have recently reported the purification of the native sodium- and chloride-coupled glycine transporter from pig brain stem (López-Corcuera, B. Vázquez, J., and Aragón, C. (1991) J. Biol. Chem. 266, 24809-24814). This preparation is essentially homogeneous and contains a unique 100-kDa polypeptide based on electrophoretic migration under denaturing conditions. In this paper we report the hydrodynamic characterization of the native transporter, solubilized in two different detergents, as well as the immunological identification of the protein. On the basis of results obtained from size-exclusion chromatography, we calculated the Stokes radii of transporter 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) and transporter-cholate complexes to be 5.5 and 6.0 nm, respectively. In addition, from H2O/D2O sucrose density gradient sedimentation analysis, we calculated the molecular weight of protein-detergent complexes to be 115,000 and 160,000 in CHAPS and cholate, respectively, and the molecular weight of the protein moiety as 86,000. Finally, polyclonal antibodies raised against the 100-kDa polypeptide were found to immunoprecipitate specifically glycine transport activity. Taken together, the results reported herein corroborate the identity of the 100-kDa band as the sodium- and chloride-coupled glycine transporter and also suggest that in its native state the transporter is a monomeric protein.

A rat brain cDNA encoding the neurotransmitter transporter with an unusual structure.

A rat cDNA clone encoding the novel membrane protein of the neurotransmitter transporters family was cloned and sequenced. The cDNA was identified as a transcript of the gene NTT4 of which a partial genomic clone was previously sequenced. Alignment of the amino acid sequence of NTT4 with other members of the neurotransmitter transporter family revealed a marked deviation from the conserved structure of all other members of the family. The largest extracellular loop with a potential glycosylation site was identified between membrane segments 7 and 8. The protein retains the common glycosylated loop between transmembrane helices 3 and 4 in all members of the family. The transcript of NTT4 was found exclusively in the central nervous system and is more abundant in the cerebellum and the cerebral cortex.

Cloning and expression of a cDNA encoding the transporter of taurine and beta-alanine in mouse brain.

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.