Expression of a mouse brain cDNA encoding novel gamma-aminobutyric acid transporter

THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 267, No. 25, Issue of September 5,pp. 17491-17493,1992 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A

Communication Expression of a Mouse Brain cDNA Encoding Novel ?-Aminobutyric Acid Transporter* (Received for publication, June 12, 1992) Beatriz Lopez-Corcuera, Qing-Rong Liu, Sreekala Mandiyan, Hannah Nelson, and Nathan Nelson3 From the Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110

A nipecotic acid-resistant y-aminobutyric acid (GABA) transporter was cloned from a mouse brain cDNA library. The 2.3-kilobase cDNA clone contains an open reading frame of1842 nucleotides encoding a protein of 614 amino acids. The predicted amino acid sequence indicates itis 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 BGTl which was exclusively expressed in thekidney. 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 K,,, of 79 FM for GABA uptake which is about 10-fold higherthan that of the high affinity GABA transporter (GATl). The pharmacology of GAT2 is different from that of GATl because of lack of inhibition by guvacine and nipecotic acid and sensitivity to high concentrations of betaine and B-alanine. GAT2 transports betaine with a K,,, of about 200 WM, but nosignificant transport of B-alanine could be detected. The presence of mRNA encoding GAT2 in partsof the brain suggests it is a neurotransmitter transporter.

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 (1-7). Recently two pharmacologically distinct GABA transporters were identified in plasma membrane vesicles and reconstituted preparations from rat brain (8). Both transporters were reported to be sodium-dependent and tohave a high affinity for GABA. For some time the GABA transporter in rat brain was thought to be primarily neuronal (9). Recently it was demonstrated that glial cells contain sodium-dependent GABA transporters (7,

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide seqwncefs) reported in this paper has been submitted to theGenBankTM/EMBL Data Bank with accessionnumber(s) M97632. $ To whom all correspondence should be addressed. The abbreviationsused are: GABA, y-aminobutyric acid; kb, kilobase(s).

8,lO). A cDNA encoding neuronal high affinity GABA transporter was cloned from rat (11), human (12),and mouse brain (13). The expressed cDNA exhibited specific sodium-dependent GABA uptake with a K,,, of about 7p~ (11,13).Nipecotic acid at 10-100 ~ L Minhibited GABA uptake into oocytes, and transport activity was not sensitive to ,&alanine. Inthis communication we report on the cloning and expression of cDNA encoding a nipecotic acid-resistant GABA transporter of mouse brain. MATERIALS AND METHODS

Library Screening-A mouse neonatal brain cDNA library, cloned into the EcoRI-XhoI site of the Uni-ZAP XR cloning vector, was obtained from Stratagene. The library was screened with a 3ZP-labeled 5’ end of the genomic clone NTT5 recently isolated in our laboratory (13). The library was screenedunder high stringencyconditions (hybridization at 65 “C in 5% bloto and washing at 50 “C in 0.1 X SSC containing 1% sodium dodecyl sulfate).Positive clones were verified by dot blot and Southernanalysis (14). Nucleotide sequences were aligned and analyzed using DNAstar or GCG software. A positive clone containing a DNA insert of about 2.3 kb was sequenced in both directions using oligonucleotide primers. The cDNA sequence was found to be identical to theoverlapping exon sequence of the genomic clone NTT5 (13). Northern Blots-RNA was prepared from various parts of the mouse brain as well as liver and kidney using an RNA isolation kit obtained from Stratagene. About 40 pg of total RNA was applied to each lane of the RNA gel. Staining with ethidium bromide showed similar amounts of rRNA in each lane. Hybridization with cDNA encoding the proteolipid of the vacuolar H+-ATPase also verified the equivalent amounts of mRNA in each lane (12). The hybridization and washing of the blots were carried out under high stringency. Expression of GAT2 in Xenopus Oocytes-Following linearization of the plasmid by XhoI, RNA was synthesized using Ta-RNA polymerase obtained from Stratagene. Thesynthetic RNA was injected into Xenopus oocytes, and the uptake ofGABA and other amino acids was assayed as previously described (13). RESULTS A N D DISCUSSION

Several positive clones were identified in every screening of the mouse brain library indicating the transcriptof the NTT5 gene is quite abundant in the brain. Fig. 1 shows the nucleotide and the predicted amino acid sequences of one of the cDNA clones (designated GAT2) that contained an open reading frame encoding aneurotransmitter transporter of about 70 kDa. The assigned initiator methionine is the first methionine in thesequence with the initiation site consensus sequence (15). The open reading frame can be translated into a protein of 69,667 Da containing 614 amino acids. A hydropathy plot suggests 12 transmembrane helices and a general structure similar to that proposed for the GABA transporter (GAT1) as well as other neurotransmitter transporters (1121). Two potential glycosylation sites are present on the large hydrophilic loop which is present on the outer surface of the cell membrane between the third andfourth transmembrane helices according to thepredicted structure (11). Alignment of the predicted amino acid sequence of GAT2 showed about 45% identity with noradrenaline,serotonin, and dopamine transporters (16-21), 43% identity with the mouse glycine transporter (22), 49% identity with the mouse high affinity GABA transporter (ll),and 88% identity with the betaine transporter(BGT1) recently cloned from the canine kidney library (23). The high degree of homology between the amino acid sequences of the canine transporter

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