title: An aspartate residue in the external vestibule of GLYT2 (glycine transporter 2) controls cation access and transport coupling.
abstract: Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+-Cl--glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes.
authors: Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain.
authors: Gonzalo
authors: G
authors: Pérez-Siles
authors: Enrique
authors: E
authors: Núñez
authors: Antonio
authors: A
authors: Morreale
authors: Esperanza
authors: E
authors: Jiménez
authors: Alejandra
authors: A
authors: Leo-Macías
authors: Guillermo
authors: G
authors: Pita
authors: Francesca
authors: F
authors: Cherubino
authors: Rachele
authors: R
authors: Sangaletti
authors: Elena
authors: E
authors: Bossi
authors: Angel R
authors: AR
authors: Ortíz
authors: Carmen
authors: C
authors: Aragón
authors: Beatriz
authors: B
authors: López-Corcuera
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D019556:COS Cells; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D055724:Electrophysiological Phenomena; D005260:Female; D050487:Glycine Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D013141:Spiro Compounds / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D050487:Glycine Plasma Membrane Transport Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; C493269:Slc6a5 protein, rat; C493266:Slc6a9 protein, rat; D013141:Spiro Compounds; C493992:lihouidine; D001224:Aspartic Acid; D012964:Sodium