title: A hydrophobic domain in glutamate transporters forms an extracellular helix associated with the permeation pathway for substrates.
abstract: Recent work has shown that cysteine residues introduced into domain 10, a highly hydrophobic segment in the excitatory amino acid transporter 1, react readily when hydrophilic sulfhydryl-modifying reagents are applied extracellularly. To investigate the functional contributions of this region, we mutated each residue in domain 10 (Ala(446)-Gly(459)) to cysteine and assessed the transport kinetics and inhibitor sensitivities of the mutant carriers. Modification of the introduced sulfhydryl group with membrane-impermeant methanethiosulfonate derivatives inhibited substrate transport by all but one functional cysteine mutant. Substrates and/or non-transported inhibitors block thiol modification of most mutants within this region, implying that access to the domain becomes restricted as a consequence of the binding of substrates and substrate analogs. An examination of the temperature dependence of substrate protection for one mutant (I453C) indicates that substrates prevent modification at a step prior to the large conformational changes associated with translocation. When superimposed on a helical model, mutants with similar attributes are positioned in close proximity. Our data are consistent with a model in which domain 10 exists as an alpha-helix at an aqueous interface of the translocation pathway, which can be directly occluded by substrates and inhibitors at an early step in the transport cycle.
authors.affiliation: Vollum Institute, Howard Hughes Medical Institute, Oregon Health & Sciences University, Portland, Oregon 97201, USA.
authors.forename: Barbara H
authors.initials: BH
authors.lastname: Leighton
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Seal
authors.forename: Keiko
authors.initials: K
authors.lastname: Shimamoto
authors.forename: Susan G
authors.initials: SG
authors.lastname: Amara
mesh_terms: D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D027341:Excitatory Amino Acid Transporter 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
chemical_list: D027341:Excitatory Amino Acid Transporter 1