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.

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.

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.