Abstract
A nipecotic acid-resistant gamma-aminobutyric acid (GABA) transporter was cloned from a mouse brain cDNA library. The 2.3-kilobase cDNA clone contains an open reading frame of 1842 nucleotides encoding a protein of 614 amino acids. The predicted amino acid sequence indicates it is a member of the gene family of the sodium-dependent neurotransmitter transporters. The new GABA transporter, named GAT2, is highly homologous to the betaine transporter (BGT1) cloned from canine kidney. However, GAT2 expression in the brain distinguished it from BGT1 which was exclusively expressed in the kidney. The transcripts of GAT2 were found in the cerebral cortex, cerebellum, and brainstem as well as in kidney. Expression of GAT2 in Xenopus oocytes revealed a Km of 79 microM for GABA uptake which is about 10-fold higher than that of the high affinity GABA transporter (GAT1). The pharmacology of GAT2 is different from that of GAT1 because of lack of inhibition by guvacine and nipecotic acid and sensitivity to high concentrations of betaine and beta-alanine. GAT2 transports betaine with a Km of about 200 microM, but no significant transport of beta-alanine could be detected. The presence of mRNA encoding GAT2 in parts of the brain suggests it is a neurotransmitter transporter.
Highlights
Library Screening-A mouse neonatal brain cDNA library, cloned into the EcoRI-XhoI site of the Uni-ZAP XR cloning vector, was (GABA) transporter was cloned from a mouse brain obtained from Stratagene
It is assumed that the synaptic transmission of y-aminobutyric acid (GABA)’ is terminated by transportation of the amino acid into nearby cellular structures [1,2]
Studies of GABA uptake into rat cerebral cortex slices, isolated cells, and synaptosomes revealed a multitude of transporters with K, values ranging from 2 p M to 2 mM [1,2,3,4,5,6,7]
Summary
Library Screening-A mouse neonatal brain cDNA library, cloned into the EcoRI-XhoI site of the Uni-ZAP XR cloning vector, was (GABA) transporter was cloned from a mouse brain obtained from Stratagene. The predicted amino acid (hybridization at 65 “C in 5% bloto and washing at 50 “C in 0.1 X sequence indicates itis a member of the gene familyof SSC containing 1% sodium dodecyl sulfate).Positive clones were the sodium-dependent neurotransmitter transporters. About 40 pgof total RNA was applied to GAT2 in Xenopus oocytes revealed a K,,, of 79 FM for GABA uptake which is about 10-fold higherthan that of the high affinity GABA transporter (GATl). Hybridization with cDNA encoding the proteolipid of the vacuolar H+-ATPasealso verified the equivalent amounts of mRNA in each lane [12]. Expression of GAT2 in Xenopus Oocytes-Following linearization of the plasmid by XhoI, RNA was synthesized using Ta-RNA polymerase obtainedfrom Stratagene. Thesynthetic RNA was injected into about 200 WM, but nosignificant transport of B-alanine Xenopus oocytes, and the uptake ofGABA and other amino acids could be detected. GAT2 in partsof the brain suggests it is a neurotransmitter transporter
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