title: Sulfhydryl modification of V449C in the glutamate transporter EAAT1 abolishes substrate transport but not the substrate-gated anion conductance.
abstract: Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. EC(50)s for L-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the EC(50) for L-glutamate to activate the anion conductance, without affecting the EC(50) for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation.
authors.affiliation: Vollum Institute, Howard Hughes Medical Institute, Oregon Health Sciences University L-474, Portland, OR 97201, USA.
authors.forename: R P
authors.initials: RP
authors.lastname: Seal
authors.forename: Y
authors.initials: Y
authors.lastname: Shigeri
authors.forename: S
authors.initials: S
authors.lastname: Eliasof
authors.forename: B H
authors.initials: BH
authors.lastname: Leighton
authors.forename: S G
authors.initials: SG
authors.lastname: Amara
mesh_terms: D000838:Anions; D001692:Biological Transport; D027341:Excitatory Amino Acid Transporter 1 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D000838:Anions; D027341:Excitatory Amino Acid Transporter 1; D013438:Sulfhydryl Compounds