title: Proximity of transmembrane domains 1 and 3 of the gamma-aminobutyric acid transporter GAT-1 inferred from paired cysteine mutagenesis.
abstract: GAT-1 is a sodium- and chloride-dependent gamma-aminobutyric acid transporter and is the first identified member of a family of transporters that maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. Because transmembrane domains 1 and 3 contain amino acid residues important for transport activity, we hypothesized that these domains may participate in the formation of the binding pocket of the transporter. Pairwise substitutions have been introduced in several predicted transmembrane domains and in the first extracellular loop of GAT-1. In the double mutant W68C/I143C, in which the cysteines were introduced at locations at the extracellular part of transmembrane domains 1 and 3, respectively, approximately 70% inhibition of transport was observed by cadmium with an IC50 of approximately 10 microm. This inhibition was not observed in the corresponding single mutants and also not in > 10 other double mutants, except for V67C/I143C, where the half-maximal effect was obtained at approximately 50 microm. The inhibition by cadmium was only observed when the cysteine pairs were introduced in the same polypeptide. Our results suggest that transmembrane domains 1 and 3 come in close proximity within the transporter monomer.
authors.affiliation: Department of Biochemistry, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel.
authors.forename: Elia
authors.initials: E
authors.lastname: Zomot
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Zhou
authors.forename: Baruch I
authors.initials: BI
authors.lastname: Kanner
mesh_terms: D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002104:Cadmium / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics; D050485:GABA Plasma Membrane Transport Proteins; D006367:HeLa Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D050485:GABA Plasma Membrane Transport Proteins; D026901:Membrane Transport Proteins; C493413:SLC6A1 protein, human; D013438:Sulfhydryl Compounds; D002104:Cadmium; D005680:gamma-Aminobutyric Acid; D003545:Cysteine