title: The aromatic and charge pairs of the thin extracellular gate of the γ-aminobutyric acid transporter GAT-1 are differently impacted by mutation.
abstract: GAT-1 is a sodium- and chloride-coupled GABA transporter and a member of the neurotransmitter:sodium:symporters, which are crucial for synaptic transmission. The structure of bacterial homologue LeuT shows a thin extracellular gate consisting of a charge and an aromatic pair. Here we addressed the question of whether mutation of the aromatic and charge pair residues of GAT-1 has similar consequences. In contrast to charge pair mutants, significant radioactive GABA transport was retained by mutants of the aromatic pair residue Phe-294. Moreover, the magnitude of maximal transport currents induced by GABA by these mutants was comparable with those by wild type GAT-1. However, the apparent affinity of the nonconserved mutants for GABA was reduced up to 20-fold relative to wild type. The voltage dependence of the sodium-dependent transient currents of the Phe-294 mutants was similar to that of the wild type. On the other hand, the conserved charge pair mutant D451E exhibited a right-shifted voltage dependence, indicating an increased apparent affinity for sodium. In further contrast to D451E, whereas the extracellular aqueous accessibility of an endogenous cysteine residue to a membrane-impermeant sulfhydryl reagent was increased relative to wild type, this was not the case for the aromatic pair mutants. Our data indicate that, in contrast to the charge pair, the aromatic pair is not essential for gating. Instead they are compatible with the idea that they serve to diminish dissociation of the substrate from the binding pocket.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel.
authors.forename: Oshrat
authors.initials: O
authors.lastname: Dayan
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel.
authors.forename: Assaf
authors.initials: A
authors.lastname: Ben-Yona
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel baruch.kanner@mail.huji.ac.il.
authors.forename: Baruch I
authors.initials: BI
authors.lastname: Kanner
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism; D001692:Biological Transport; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials; D008958:Models, Molecular; D009154:Mutation; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D010649:Phenylalanine / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000737:chemistry* / Q000378:metabolism
chemical_list: D050485:GABA Plasma Membrane Transport Proteins; D011994:Recombinant Proteins; C493413:SLC6A1 protein, human; D001224:Aspartic Acid; D018698:Glutamic Acid; D010649:Phenylalanine; D005680:gamma-Aminobutyric Acid; D012964:Sodium
keywords: Gating; Kinetics; Neurotransmitter Transport; Transmembrane Domain; Transport