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...
The Journal of biological chemistry, 1993
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...
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.
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.
Neuroscience Letters, 1998
We have identified two alternative 5′ ends for the GLYT2 glycine transporter in rat brain DNA by using rapid amplification of cDNA ends (RACE) analysis. Study of the genomic DNA revealed that the isoform diversity is generated by alternative use of exons Ia or Ib, respectively. Upon translation, the mRNA corresponding to the novel isoform GLYT2b would yield a protein five amino acids longer than the previously characterized isoform GLYT2a. Both forms display similar regional distribution and kinetics characteristics. However, whereas GLYT2a is able to actively accumulate glycine into transfected COS cells, GLYT2b seems only to exchange (or release) glycine.