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J. Biol. Chem., Vol. 282, Issue 27, 19331-19341, July 6, 2007

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Cloning and Characterization of a Functional Human -Aminobutyric Acid (GABA) Transporter, Human GAT-2^*

Bolette Christiansen, Anne-Kristine Meinild, Anders A. Jensen, and Hans Braüner-Osborne¹

From the Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences and Department of Molecular Biology, Faculty of Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark

Plasma membrane -aminobutyric acid (GABA) transporters act to terminate GABA neurotransmission in the mammalian brain. Intriguingly four distinct GABA transporters have been cloned from rat and mouse, whereas only three functional homologs of these transporters have been cloned from human. The aim of this study therefore was to search for this fourth missing human transporter. Using a bioinformatics approach, we successfully identified and cloned the full-length cDNA of a so far uncharacterized human GABA transporter (GAT). The predicted protein displays high sequence similarity to rat GAT-2 and mouse GAT3, and in accordance with the nomenclature for rat GABA transporters, we therefore refer to the transporter as human GAT-2. We used electrophysiological and cell-based methods to demonstrate that this protein is a functional transporter of GABA. The transport was saturable and dependent on both Na⁺ and Cl^–. Pharmacologically the transporter is distinct from the other human GABA transporters and similar to rat GAT-2 and mouse GAT3 with high sensitivity toward GABA and β-alanine. Furthermore the GABA transport inhibitor (S)-SNAP-5114 displayed some inhibitory activity at the transporter. Expression analysis by reverse transcription-PCR showed that GAT-2 mRNA is present in human brain, kidney, lung, and testis. The finding of the human GAT-2 demonstrates for the first time that the four plasma membrane GABA transporters identified in several mammalian species are all conserved in human. Furthermore the availability of human GAT-2 enables the use of all human clones of the GABA transporters in drug development programs and functional characterization of novel inhibitors of GABA transport.

Received for publication, March 12, 2007 , and in revised form, May 10, 2007.

^* This work was supported by the Lundbeck Foundation, the Novo Nordisk Foundation, the Carlsberg Foundation, and the Danish Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. Tel.: 45-3533-6518; Fax: 45-3533-6040; E-mail: hbo{at}molpharm.net.

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