TY - JOUR

T1 - Cloning and characterization of a functional human ¿-aminobutyric acid (GABA) transporter, human GAT-2

AU - Christiansen, Bolette

AU - Meinild, Anne-Kristine

AU - Jensen, Anders A.

AU - Bräuner-Osborne, Hans

PY - 2007

Y1 - 2007

N2 - Plasma membrane gamma-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 beta-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.

AB - Plasma membrane gamma-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 beta-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.

KW - Animals

KW - Anisoles

KW - Biological Transport, Active

KW - Cell Line, Transformed

KW - Cell Membrane

KW - Chlorides

KW - Cloning, Molecular

KW - DNA, Complementary

KW - GABA Plasma Membrane Transport Proteins

KW - GABA Uptake Inhibitors

KW - Gene Expression Regulation

KW - Humans

KW - Mice

KW - Nipecotic Acids

KW - Organ Specificity

KW - RNA, Messenger

KW - Rats

KW - Sequence Homology, Amino Acid

KW - Sodium

KW - gamma-Aminobutyric Acid

U2 - 10.1074/jbc.M702111200

DO - 10.1074/jbc.M702111200

M3 - Journal article

C2 - 17502375

VL - 282

SP - 19331

EP - 19341

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 27

ER -