Cloning and expression of a glycine transporter from mouse brain (original) (raw)
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Glycine transporters are differentially expressed among CNS cells
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1995
Glycine is the major inhibitory neurotransmitter in the spinal cord and brainstem and is also required for the activation of NMDA receptors. The extracellular concentration of this neuroactive amino acid is regulated by at least two glycine transporters (GLYT1 and GLYT2). To study the localization and properties of these proteins, sequence-specific antibodies against the cloned glycine transporters have been raised. Immunoblots show that the 50-70 kDa band corresponding to GLYT1 is expressed at the highest concentrations in the spinal cord, brainstem, diencephalon, and retina, and, in a lesser degree, to the olfactory bulb and brain hemispheres, whereas it is not detected in peripheral tissues. Pre-embedding light and electron microscopic immunocytochemistry show that GLYT1 is expressed in glial cells around both glycinergic and nonglycinergic neurons except in the retina, where it is expressed by amacrine neurons, but not by glia. The expression of a 90-110 kDa band corresponding t...
Differential Properties of Two Stably Expressed Brain-Specific Glycine Transporters
Journal of Neurochemistry, 2002
Clonal cell lines stably expressing the glial glycine transporter lb (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 Nadependence 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 Naõf GLYT2 than GLYT1b is suggested. One CL is required for GLYT1b and GLYT2 transport cycles, although GLYT1 b 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. Key Words: Glycine transporters-Brain-Expression.
Enhancement of the NMDA receptor function by reduction of glycine transporter-1 expression
Neuroscience Letters, 2004
The occupation of the glycine binding-site is a prerequisite for NMDA receptor activation by glutamate. To analyze the regulation of NMDA receptor function by the glycine transporter 1 (GlyT1), we generated heterozygous constitutive GlyT1 knockout mice (GlyT1tm1.1 +/− ). These animals were fully viable. Using a newly generated antibody, the pattern of GlyT1 expression in brain was found to be unaltered in the mutants while the level of expression was strongly reduced in all brain regions, as shown immunohistochemically. In hippocampal slices the ratio of the peak amplitude of NMDA and AMPA receptor evoked excitatory postsynaptic currents (EPSCs), recorded in CA1 pyramidal cells, was significantly enhanced by 36% in Glyt1tm1.1 +/− compared to wild-type slices. The frequency and amplitude of AMPA miniature events in Glyt1tm1.1 +/− mice were indistinguishable from those recorded in wild type. These results provide proof that the NMDA receptor function is enhanced by a reduction of GlyT1 expression. Thus, GlyT1 function is a controlling factor for an enhancement of the NMDA receptor response. These findings are of relevance for the development of GlyT1 inhibitory drugs.
Modulation of N -methyl- d -aspartate receptor function by glycine transport
Proceedings of the National Academy of Sciences, 1998
The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N -methyl- d -aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch–clamp recordings of hippocampal pyramidal neurons, in vitro , demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 μM glycine, a concentration similar to that in the cerebrospinal fluid in vivo , thereby supporting the hypothesis that the GLYT1 maintains subsa...
Journal of Neurochemistry, 2002
We studied by immunocytochemical localization, the glycine neurotransmitter transporter (GLYT2) in mouse brain, using polyclonal antibodies raised against recombinant N-terminus and loop fusion proteins. Western analysis and immunocytochemistry of mouse brain frozen sections revealed caudal-rostrai gradient of GLYT2 distribution with massive accumulation in the spinal cord, brainstem, and less in the cerebellum. Immunereactivity was detected in processes with varicosities but not cell bodies. A correlation was observed between the pattern we obtained and previously reported strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission accompanying the glycine receptor at the classic inhibitory system in the hindbrain.
Biochemical and Biophysical Research Communications, 2012
Glycine serves as a neurotransmitter in spinal cord and brain stem, where it activates inhibitory glycine receptors. In addition, it serves as an essential co-agonist of excitatory N-methyl-d-aspartate receptors. In the central nervous system, extracellular glycine concentrations are regulated by two specific glycine transporters (GlyTs), GlyT1 and GlyT2. Here, we determined the relative transport activities and protein levels of GlyT1 and GlyT2 in membrane preparations from mouse brain stem and spinal cord at different developmental stages. We report that early postnatally (up to postnatal day P5) GlyT1 is the predominant transporter isoform responsible for a major fraction of the GlyT-mediated [(3)H]glycine uptake. At later stages (≥ P10), however, the transport activity and expression of GlyT2 increases, and in membrane fractions from adult mice both GlyTs contribute about equally to glycine uptake. These alterations in the activities and expression profiles of the GlyTs suggest that the contributions of GlyT1 and GlyT2 to the regulation of extracellular glycine concentrations at glycinergic synapses changes during development.
Localization of the GLYT1 Glycine Transporter at Glutamatergic Synapses in the Rat Brain
Cerebral Cortex, 2005
In this study, we present evidence that a glycine transporter, GLYT1, is expressed in neurons and that it is associated with glutamatergic synapses. Despite the presence of GLYT1 mRNA in both glial cells and in glutamatergic neurons, previous studies have mainly localized GLYT1 immunoreactivity to glial cells in the caudal regions of the nervous system. However, using novel sequence specific antibodies, we have identified GLYT1 not only in glia, but also in neurons. The immunostaining of neuronal elements could best be appreciated in forebrain areas such as the neocortex or the hippocampus, and it was found in fibers, terminal boutons and in some dendrites. Double labeling confocal microscopy with the glutamatergic marker vGLUT1 revealed an enrichment of GLYT1 in a subpopulation of glutamatergic terminals. Moreover, through electron microscopy, we observed an enrichment of GLYT1 in both the presynaptic and the postsynaptic aspects of putative glutamatergic terminals that established asymmetric synapses. In addition, we demonstrated that GLYT1 was physically associated with the NMDA receptor in a biochemical assay. In conclusion, the close spatial association of GLYT1 and glutamatergic synapses strongly supports a role for this protein in neurotransmission mediated by NMDA receptors in the forebrain, and perhaps in other regions of the CNS.