A cDNA clone encoding a glycine transporter has been isolated from rat brain by a combined PCR and plaque-hybridization strategy. mRNA synthesized from this clone (designated GLYT1) directs the expression of sodiumand chloride-dependent, high-affinity uptake of [3H]glycine by Xenopus oocytes.[3H]Glycine transport mediated by clone GLYT1 is blocked by sarcosine but is not blocked by methylaminoisobutyric acid or L-alanine, a substrate specificity similar to that described for a previously identified glycine-uptake system called system Gly. In situ hybridization reveals that GLYT1 is prominently expressed in the cervical spinal cord and brainstem, two regions of the central nervous system where glycine is a putative neurotransmitter. GLYT1 is also strongly expressed in the cerebellum and olfactory bulb and is expressed at lower levels in other brain regions. The open reading frame of the GLYT1 cDNA predicts a protein containing 633 amino acids with a molecular mass of =70 kDa. The primary structure and hydropathicity profile of GLYT1 protein reveal that this protein is a member of the sodium-and chloride-dependent superfamily of transporters that utilize neurotransmitters and related substances as substrates.Termination of synaptic activity is thought to occur through removal of neurotransmitter from the synaptic cleft by ioncoupled, high-affinity neurotransmitter transport proteins (neurotransmitter transporters) located in neuronal and glial plasma membranes. Drugs that block the action of these transporters can modulate neural function, probably by increasing the duration of neurotransmitter action. Some neurotransmitter transporters, such as those for the monoamines, are the sites of action for clinically important drugs (for example, antidepressants) (1), as well as drugs of abuse (for example, cocaine) (2). Other neurotransmitter transporters, such as those for the inhibitory amino acid y-aminobutyric acid (GABA), are potential targets for drugs with anticonvulsant properties (3). Therefore, an understanding of the structure and function of neitrotransmitter transporters may lead to a better understanding of synaptic regulation and could also provide a rational basis for the development of additional, more-specific anti-transporter drugs.The recent isolation of cDNA clones encoding transporters for GABA (4), the monoamines norepinephrine, dopamine, and serotonin (5-10), and a nonneurotransmitter, betaine (11), has revealed that these carriers comprise a superfamily of homologous proteins. Predictions of membrane topology suggest that these transporters all contain =12 membranespanning domains, a structural motif shared by a number of carriers that, however, share little mutual sequence homology. The amino acid-sequence identity between the known members of the neurotransmitter transporter superfamily ranges from "-44% for the GABA transporter (GAT1) versus the norepinephrine transporter (NET1) to =64% for NET1 versus the dopamine transporter (DAT1), indicating that subfamilies exist within the superf...
Gamma-aminobutyric acid (GABA) plasma membrane transporters influence synaptic transmission by high-affinity, Na(+)-dependent transport processes. The cDNA clone, GAT-1, encodes a high-affinity Na(+)- and Cl(-)-dependent GABA plasma membrane transporter, which has kinetic and pharmacological properties similar to those of high-affinity GABA uptake systems associated with neurons. The present study evaluates the distribution and cellular localization of this putative neuronal GABA transporter by RNA blot hybridization and in situ hybridization histochemistry in the rat retina. Northern blot hybridization analysis of total retinal and cerebellar RNA extracts demonstrated a single band of hybridization at 4.2 kilobases. GABA transporter mRNA is expressed by numerous cells that are distributed to the proximal inner nuclear layer and the ganglion cell layer and by a few cells located in the inner plexiform layer. Double label studies combining the retrograde transport of the fluorescent dye Fluorogold from the superior colliculus to identify ganglion cells and in situ hybridization histochemistry demonstrated that most GAT-1 mRNA-containing cells in the ganglion cell layer are displaced amacrine cells, although some ganglion cells containing GAT-1 mRNA were visualized. In freshly dissociated retinal cell preparations, the GAT-1 RNA signal is strong in neurons and weak to moderate in specialized glial cells called Müller cells. Müller cells were identified by both their morphology and the presence of the selective Müller cell marker cellular retinaldehyde-binding protein. Only background labeling is seen with the sense GAT-1 RNA probe in both tissue sections and dissociated retinal cell preparations. These findings demonstrate that GAT-1 mRNA is expressed in both the retina and brain. In the retina, this transporter is predominantly localized to amacrine, displaced amacrine and interplexiform cells, and some ganglion cells. This transporter mRNA is also expressed by Müller cells but at a lower level than by neurons. These observations indicate that GABA transport by GAT-1 plasma membrane transporters in the retina is mediated by both neurons and glia cells.
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