Cloning and Expression of a Rat Brain GABA Transporter

Abstract

A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C

“…The cDNA coding for GAT1 was isolated by screening a AZAPII rat spinal cord library with a PCR fragment containing nucleotide positions 46-1773 of the GAT1 sequence [13]. This resulted in the isolation of a full-length cDNA encompassing all nucleotides of the published GATl-coding sequence and 150 and 2104 bp of the 5' and 3' untranslated regions, respectively [13].…”

“…This resulted in the isolation of a full-length cDNA encompassing all nucleotides of the published GATl-coding sequence and 150 and 2104 bp of the 5' and 3' untranslated regions, respectively [13]. For heterologous expression, the coding sequence of GAT1, including 150 bp of 5' untranslated sequence and 165 bp of 3' untranslated sequence, was subcloned into the NotI/HpaI sites of the eucaryotic expression vector pCIS [14].…”

Reduced Anxiety and Depression-Like Behaviors in Mice Lacking GABA Transporter Subtype 1

“…Molecular cloning studies have identified multiple GABA transporter subtypes, including GAT1, GAT2, GAT3, and GAT4. GAT1 is the major subtype present at both synaptic and extrasynaptic sites in the brain (Chiu et al, 2002;Guastella et al, 1990). Our previous work had demonstrated that the GAT1 gene knockout mice have decreased ethanol aversion and ethanol reward, and insensitivity to both the sedative/ hypnotic and the motor stimulant effects of ethanol (Cai et al, 2006).…”

GABA transporter 1 transcriptional starting site exhibiting tissue specific difference

“…GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and adult mouse brain Expression of GAT1 was detected in rat cerebrum and cerebellum [1]. By means of 5 RACE using primer P1, P2 (indicated in Fig 2) and sequence analysis, we found that the GAT1 transcriptional starting site in neonatal mouse cerebral cortex (GenBank No.…”

“…GAT1 was the first cloned member of neurotransmitter transporters superfamily [1], and soon, other three subtypes (GAT2-4) were subsequently cloned. Since GABA is the predominant inhibitory neurotransmitter in CNS, abnormality of GATs has a direct relationship with certain kinds of nervous disorders, such as epilepsy and cognitive deterioration.…”