title: Conformationally sensitive proximity of extracellular loops 2 and 4 of the γ-aminobutyric acid (GABA) transporter GAT-1 inferred from paired cysteine mutagenesis.
abstract: The sodium- and chloride-coupled GABA transporter GAT-1 is a member of the neurotransmitter:sodium:symporters, which are crucial for synaptic transmission. Structural work on the bacterial homologue LeuT suggests that extracellular loop 4 closes the extracellular solvent pathway when the transporter becomes inward-facing. To test whether this model can be extrapolated to GAT-1, cysteine residues were introduced at positions 359 and 448 of extracellular loop 4 and transmembrane helix 10, respectively. Treatment of HeLa cells, expressing the double cysteine mutant S359C/K448C with the oxidizing reagent copper(II)(1,10-phenantroline)3, resulted in a significant inhibition of [(3)H]GABA transport. However, transport by the single cysteine mutant S359C was also inhibited by the oxidant, whereas its activity was almost 4-fold stimulated by dithiothreitol. Both effects were attenuated when the conserved cysteine residues, Cys-164 and/or Cys-173, were replaced by serine. These cysteines are located in extracellular loop 2, the role of which in the structure and function of the eukaryotic neurotransmitter:sodium:symporters remains unknown. The inhibition of transport of S359C by the oxidant was markedly reduced under conditions expected to increase the proportion of inward-facing transporters, whereas the reactivity of the mutants to a membrane-impermeant sulfhydryl reagent was not conformationally sensitive. Our data suggest that extracellular loops 2 and 4 come into close proximity to each other in the outward-facing conformation of GAT-1.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel.
authors.forename: Maram
authors.initials: M
authors.lastname: Hilwi
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University Hadassah Medical School, P. O. Box 12272, 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, P. O. Box 12272, Jerusalem 91120, Israel baruch.kanner@mail.huji.ac.il.
authors.forename: Baruch I
authors.initials: BI
authors.lastname: Kanner
mesh_terms: D000818:Animals; D002794:Choline / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005942:Gluconates / Q000378:metabolism / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D009154:Mutation; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010618:Phenanthrolines / Q000737:chemistry; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D050485:GABA Plasma Membrane Transport Proteins; D005942:Gluconates; D010618:Phenanthrolines; D011994:Recombinant Proteins; C493413:SLC6A1 protein, human; C118336:copper(II)(1,10-phenanthroline)3; D005680:gamma-Aminobutyric Acid; D003545:Cysteine; D002794:Choline; C030691:gluconic acid
keywords: Conformational Change; Cysteine-mediated Cross-linking; Disulfide; GABA; Neurotransmitter Transport; Sulfhydryl