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

Abstract

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 ␤-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.

High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach

“…S5 and S6). These values are in agreement with previous uptake inhibition data for GABAergic molecules (4,9). Six of the identified ligands (homotaurine, baclofen, gabapentin, lorazepam, pyridoxal phosphate, and ZAPA) are chemically novel GAT-2 ligands (Fig.…”

“…S4). The functionality of the assay was demonstrated by comparing kinetic parameters with those previously reported for GAT-2 in published data (4,8,9) and by competitive inhibition of 3 H-GABA uptake with unlabeled GABA in addition to the known GAT-2 inhibitors ␤-alanine and desipramine.…”

“…In particular, we calculated the enrichment for the known ligands among the top scoring decoy compounds, generated by the Directory of Useful Decoys protocol (32,43). 11 GAT-2 ligands were collected from the literature (4,9), as well as the UniProt (44) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (45) databases. For each known ligand and 36 Directory of Useful Decoys-generated decoys, the best docking pose was com-puted.…”

“…These drugs increase the concentration of GABA in the synaptic cleft by inhibiting GAT-1-or GAT-3-mediated GABA reuptake. The GABA transporter 2 (GAT-2, SLC6A13) is primarily expressed in the liver, kidney, and other peripheral tissues such as the testis, retina, and lungs (4,8,9). Thus, GAT-2 might be physiologically important for regulating key peripheral GABAergic mechanisms such as those associated with asthma and diabetes (6,7).…”

Early developmental alterations in GABAergic protein expression in fragile X knockout mice

“…Neurotransmission is terminated through the reuptake of GABA into presynaptic nerve terminals in a recycling process mediated by GABA transporters (GATs), which also co-transport for sodium and chloride ions (Takayama and Inoue, 2005). Four distinct GABA transporters, GAT-1, BGT-1, GAT-2, and GAT-3 have been identified in several mammalian species (Christiansen et al, 2007). GABA uptake can also occur to surrounding glia where GABA transaminase (GABA-T) converts GABA into succinic semialdehyde.…”