title: A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation.
abstract: To investigate the structural determinants underlying transport by the glutamate transporter EAAT1, we mutated each of 24 highly conserved residues (P392 to Q415) to cysteine. A majority of these substituted cysteines react with the sulfhydryl-modifying reagent MTSEA, suggesting that they reside in an aqueous environment. The impermeant reagents MTSES and MTSET react with residues at each end of the domain (A395C and A414C), supporting a model that places these residues near the extracellular surface. Substrates and inhibitors block the reaction between MTS derivatives and A395C, and the cosubstrate, sodium, slows reaction of MTSEA with Y405C and E406C. From these results, we propose that this domain forms a reentrant membrane loop at the cell surface and may comprise part of the translocation pore for substrates and cotransported ions.
authors.affiliation: Program in Neuroscience, Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.
authors.forename: R P
authors.initials: RP
authors.lastname: Seal
authors.forename: S G
authors.initials: SG
authors.lastname: Amara
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D017124:Conserved Sequence; D003545:Cysteine; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D013816:Thermodynamics; D014162:Transfection
chemical_list: D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D011994:Recombinant Proteins; D012964:Sodium; D003545:Cysteine