title: Substrate-induced conformational changes of extracellular loop 1 in the glycine transporter GLYT2.
abstract: The neurotransmitter glycine is removed from the synaptic cleft by two Na(+)-and Cl(-)-dependent transporters, the glial (GLYT1) and neuronal (GLYT2) glycine transporters. GLYT2 lacks a conserved cysteine in the first hydrophilic loop (EL1) that is reactive to [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET) in related transporters. A chimeric GLYT2 (GLYT2a-EL1) that contains GLYT1 sequences in this region, including the relevant cysteine, was sensitive to the reagent, and its sensitivity was decreased by co-substrates. We combined cysteine-specific biotinylation to detect transporter-reagent interactions with MTSET inactivation assays and temperature dependence analysis to study the mechanism by which Cl(-), Na(+), and glycine reduce methanethiosulfonate reagent inhibition. We demonstrate a Na(+) protective effect rather than an increased susceptibility to the reagent exerted by Li(+), as reported for the serotonin transporter. The different inhibition, protection, and reactivation properties between GLYT2a-EL1 and serotonin transporter suggest that EL1 is a source of structural heterogeneity involved in the specific effect of lithium on serotonin transport. The protection by Na(+) or Cl(-) on GLYT2a-EL1 was clearly dependent on temperature, suggesting that EL1 is not involved in ion binding but is subjected to ion-induced conformational changes. Na(+) and Cl(-) were required for glycine protection, indicating the necessity of prior ion interaction with the transporter for the binding of glycine. We conclude that EL1 acts as a fluctuating hinge undergoing sequential conformational changes during the transport cycle.
authors.affiliation: Centro de Biologia Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Cientificas, 28049-Madrid, Spain.
authors.forename: B
authors.initials: B
authors.lastname: López-Corcuera
authors.forename: E
authors.initials: E
authors.lastname: Núñez
authors.forename: R
authors.initials: R
authors.lastname: Martínez-Maza
authors.forename: A
authors.initials: A
authors.lastname: Geerlings
authors.forename: C
authors.initials: C
authors.lastname: Aragón
mesh_terms: D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry*; D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D019556:COS Cells; D002713:Chlorine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D050487:Glycine Plasma Membrane Transport Proteins; D007477:Ions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000378:metabolism; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D050487:Glycine Plasma Membrane Transport Proteins; D007477:Ions; D012701:Serotonin; D002713:Chlorine; D012964:Sodium; D005998:Glycine