title: High selectivity of the γ-aminobutyric acid transporter 2 (GAT-2, SLC6A13) revealed by structure-based approach.
abstract: The solute carrier 6 (SLC6) is a family of ion-dependent transporters that mediate uptake into the cell of osmolytes such as neurotransmitters and amino acids. Four SLC6 members transport GABA, a key neurotransmitter that triggers inhibitory signaling pathways via various receptors (e.g., GABA(A)). The GABA transporters (GATs) regulate the concentration of GABA available for signaling and are thus targeted by a variety of anticonvulsant and relaxant drugs. Here, we characterize GAT-2, a transporter that plays a role in peripheral GABAergic mechanisms, by constructing comparative structural models based on crystallographic structures of the leucine transporter LeuT. Models of GAT-2 in two different conformations were constructed and experimentally validated, using site-directed mutagenesis. Computational screening of 594,166 compounds including drugs, metabolites, and fragment-like molecules from the ZINC database revealed distinct ligands for the two GAT-2 models. 31 small molecules, including high scoring compounds and molecules chemically related to known and predicted GAT-2 ligands, were experimentally tested in inhibition assays. Twelve ligands were found, six of which were chemically novel (e.g., homotaurine). Our results suggest that GAT-2 is a high selectivity/low affinity transporter that is resistant to inhibition by typical GABAergic inhibitors. Finally, we compared the binding site of GAT-2 with those of other SLC6 members, including the norepinephrine transporter and other GATs, to identify ligand specificity determinants for this family. Our combined approach may be useful for characterizing interactions between small molecules and other membrane proteins, as well as for describing substrate specificities in other protein families.
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94158, USA.
authors.forename: Avner
authors.initials: A
authors.lastname: Schlessinger
authors.forename: Matthias B
authors.initials: MB
authors.lastname: Wittwer
authors.forename: Amber
authors.initials: A
authors.lastname: Dahlin
authors.forename: Natalia
authors.initials: N
authors.lastname: Khuri
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Bonomi
authors.forename: Hao
authors.initials: H
authors.lastname: Fan
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Giacomini
authors.forename: Andrej
authors.initials: A
authors.lastname: Sali
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects; D018360:Crystallography, X-Ray; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D054852:Small Molecule Libraries; D015262:Xenobiotics / Q000737:chemistry / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000737:chemistry* / Q000378:metabolism
chemical_list: D050485:GABA Plasma Membrane Transport Proteins; D008024:Ligands; C493418:SLC6A13 protein, human; D054852:Small Molecule Libraries; D015262:Xenobiotics; D005680:gamma-Aminobutyric Acid