Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport

Neurotransmitter removal from glycine-mediated synapses relies on two sodium-driven high-affinity plasma membrane GlyTs that control neurotransmitter availability. Mostly glial GlyT1 is the main regulator of glycine synaptic levels, whereas neuronal GlyT2 promotes the recycling of synaptic glycine a...

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Detalles Bibliográficos
Autores: Benito Muñoz, Cristina, Perona Requena, Almudena, Abia, David, Santos, Helena G. dos, Núñez, Enrique, Aragón, Carmen, López Corcuera, Beatriz
Tipo de recurso: artículo
Fecha de publicación:2018
País:España
Institución:Universidad Europea (UEM)
Repositorio:ABACUS. Repositorio de Producción Científica
Idioma:inglés
OAI Identifier:oai:abacus.universidadeuropea.com:11268/8619
Acceso en línea:http://hdl.handle.net/11268/8619
Access Level:acceso abierto
Palabra clave:Neurología
Biología Molecular
Biología molecular
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spelling Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate TransportBenito Muñoz, CristinaPerona Requena, AlmudenaAbia, DavidSantos, Helena G. dosNúñez, EnriqueAragón, CarmenLópez Corcuera, BeatrizNeurologíaBiología MolecularNeurologíaBiología molecularNeurotransmitter removal from glycine-mediated synapses relies on two sodium-driven high-affinity plasma membrane GlyTs that control neurotransmitter availability. Mostly glial GlyT1 is the main regulator of glycine synaptic levels, whereas neuronal GlyT2 promotes the recycling of synaptic glycine and supplies neurotransmitter for presynaptic vesicle refilling. The GlyTs differ in sodium:glycine symport stoichiometry, showing GlyT1 a 2:1 and GlyT2 a 3:1 sodium:glycine coupling. Sodium binds to the GlyTs at two conserved Na+ sites: Na1 and Na2. The location of GlyT2 Na3 site remains unknown, although Glu650 has been involved in the coordination. Here, we have used comparative MD simulations of a GlyT2 model constructed by homology to the crystalized DAT from Drosophila melanogaster by placing the Na3 ion at two different locations. By combination of in silico and experimental data obtained by biochemical and electrophysiological analysis of GlyTs mutants, we provide evidences suggesting the GlyT2 third sodium ion is held by Glu-250 and Glu-650, within a region with robust allosteric properties involved in cation-specific sensitivity. Substitution of Glu650 in GlyT2 by the corresponding methionine in GlyT1 reduced the charge-to-flux ratio to the level of GlyT1 without producing transport uncoupling. Chloride dependence of glycine transport was almost abolished in this GlyT2 mutant but simultaneous substitution of Glu250 and Glu650 by neutral amino acids rescued chloride sensitivity, suggesting that protonation/deprotonation of Glu250 substitutes chloride function. The differential behavior of equivalent GlyT1 mutations sustains a GlyT2-specific allosteric coupling between the putative Na3 site and the chloride site.20202020-02-1820182018-01-0120182018-01-01journal articlehttp://purl.org/coar/resource_type/c_6501info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/11268/8619reponame:ABACUS. Repositorio de Producción Científicainstname:Universidad Europea (UEM)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Attribution-NonCommercial-NoDerivatives 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:abacus.universidadeuropea.com:11268/86192026-06-11T12:41:27Z
dc.title.none.fl_str_mv Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport
title Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport
spellingShingle Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport
Benito Muñoz, Cristina
Neurología
Biología Molecular
Neurología
Biología molecular
title_short Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport
title_full Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport
title_fullStr Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport
title_full_unstemmed Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport
title_sort Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport
dc.creator.none.fl_str_mv Benito Muñoz, Cristina
Perona Requena, Almudena
Abia, David
Santos, Helena G. dos
Núñez, Enrique
Aragón, Carmen
López Corcuera, Beatriz
author Benito Muñoz, Cristina
author_facet Benito Muñoz, Cristina
Perona Requena, Almudena
Abia, David
Santos, Helena G. dos
Núñez, Enrique
Aragón, Carmen
López Corcuera, Beatriz
author_role author
author2 Perona Requena, Almudena
Abia, David
Santos, Helena G. dos
Núñez, Enrique
Aragón, Carmen
López Corcuera, Beatriz
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv
dc.subject.none.fl_str_mv Neurología
Biología Molecular
Neurología
Biología molecular
topic Neurología
Biología Molecular
Neurología
Biología molecular
description Neurotransmitter removal from glycine-mediated synapses relies on two sodium-driven high-affinity plasma membrane GlyTs that control neurotransmitter availability. Mostly glial GlyT1 is the main regulator of glycine synaptic levels, whereas neuronal GlyT2 promotes the recycling of synaptic glycine and supplies neurotransmitter for presynaptic vesicle refilling. The GlyTs differ in sodium:glycine symport stoichiometry, showing GlyT1 a 2:1 and GlyT2 a 3:1 sodium:glycine coupling. Sodium binds to the GlyTs at two conserved Na+ sites: Na1 and Na2. The location of GlyT2 Na3 site remains unknown, although Glu650 has been involved in the coordination. Here, we have used comparative MD simulations of a GlyT2 model constructed by homology to the crystalized DAT from Drosophila melanogaster by placing the Na3 ion at two different locations. By combination of in silico and experimental data obtained by biochemical and electrophysiological analysis of GlyTs mutants, we provide evidences suggesting the GlyT2 third sodium ion is held by Glu-250 and Glu-650, within a region with robust allosteric properties involved in cation-specific sensitivity. Substitution of Glu650 in GlyT2 by the corresponding methionine in GlyT1 reduced the charge-to-flux ratio to the level of GlyT1 without producing transport uncoupling. Chloride dependence of glycine transport was almost abolished in this GlyT2 mutant but simultaneous substitution of Glu250 and Glu650 by neutral amino acids rescued chloride sensitivity, suggesting that protonation/deprotonation of Glu250 substitutes chloride function. The differential behavior of equivalent GlyT1 mutations sustains a GlyT2-specific allosteric coupling between the putative Na3 site and the chloride site.
publishDate 2018
dc.date.none.fl_str_mv 2018
2018-01-01
2018
2018-01-01
2020
2020-02-18
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/11268/8619
url http://hdl.handle.net/11268/8619
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution-NonCommercial-NoDerivatives 4.0 Internacional
http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution-NonCommercial-NoDerivatives 4.0 Internacional
http://creativecommons.org/licenses/by-nc-nd/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:ABACUS. Repositorio de Producción Científica
instname:Universidad Europea (UEM)
instname_str Universidad Europea (UEM)
reponame_str ABACUS. Repositorio de Producción Científica
collection ABACUS. Repositorio de Producción Científica
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,300719

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