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...

Full description

Bibliographic Details
Authors: Benito-Muñoz, Cristina, Perona Urízar, Elvira Victoria, Abia, David, Santos, Helena G. dos, Núñez, Enrique, Aragón, Carmen, López Corcuera, Beatriz
Format: article
Publication Date:2018
Country:España
Institution:Universidad Autónoma de Madrid
Repository:Biblos-e Archivo. Repositorio Institucional de la UAM
Language:English
OAI Identifier:oai:repositorio.uam.es:10486/685952
Online Access:http://hdl.handle.net/10486/685952
https://dx.doi.org/10.3389/fnmol.2018.00347
Access Level:Open access
Keyword:GlyT
Hyperekplexia
Sodium site
Neurotransmitter reuptake
SLC6
Biología y Biomedicina / Biología
id ES_bd208348e345d0aee097a2b062bac026
oai_identifier_str oai:repositorio.uam.es:10486/685952
network_acronym_str ES
network_name_str España
repository_id_str
spelling Modification of a putative third sodium site in the glycine transporter GlyT2 influences the chloride dependence of substrate transportBenito-Muñoz, CristinaPerona Urízar, Elvira VictoriaAbia, DavidSantos, Helena G. dosNúñez, EnriqueAragón, CarmenLópez Corcuera, BeatrizGlyTHyperekplexiaSodium siteNeurotransmitter reuptakeSLC6Biología y Biomedicina / BiologíaNeurotransmitter 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 siteThis work was supported by grants from the Spanish Dirección General de Investigación Científica y Técnica from the Spanish Ministerio de Economía y Competitividad (Grant Nos. SAF2014- 58045-R and SAF2017-84235-R). Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO are also acknowledged. CB-M is a recipient of a grant BES-2015-072392 from the Spanish Ministerio de Economía y Competitividad.Frontiers Media S.A.Departamento de Biología MolecularFacultad de CienciasInstituto de Investigación Sanitaria del Hospital Universitario de La Paz (IdiPAZ)Centro de Biología Molecular Severo Ochoa (CBM)20182018-09-24research articlehttp://purl.org/coar/resource_type/c_2df8fbb1VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10486/685952https://dx.doi.org/10.3389/fnmol.2018.00347reponame:Biblos-e Archivo. Repositorio Institucional de la UAMinstname:Universidad Autónoma de MadridInglésengopen accesshttp://purl.org/coar/access_right/c_abf2info:eu-repo/semantics/openAccessoai:repositorio.uam.es:10486/6859522026-06-23T12:46:27Z
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
GlyT
Hyperekplexia
Sodium site
Neurotransmitter reuptake
SLC6
Biología y Biomedicina / Biología
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 Urízar, Elvira Victoria
Abia, David
Santos, Helena G. dos
Núñez, Enrique
Aragón, Carmen
López Corcuera, Beatriz
author Benito-Muñoz, Cristina
author_facet Benito-Muñoz, Cristina
Perona Urízar, Elvira Victoria
Abia, David
Santos, Helena G. dos
Núñez, Enrique
Aragón, Carmen
López Corcuera, Beatriz
author_role author
author2 Perona Urízar, Elvira Victoria
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 Departamento de Biología Molecular
Facultad de Ciencias
Instituto de Investigación Sanitaria del Hospital Universitario de La Paz (IdiPAZ)
Centro de Biología Molecular Severo Ochoa (CBM)
dc.subject.none.fl_str_mv GlyT
Hyperekplexia
Sodium site
Neurotransmitter reuptake
SLC6
Biología y Biomedicina / Biología
topic GlyT
Hyperekplexia
Sodium site
Neurotransmitter reuptake
SLC6
Biología y Biomedicina / Biología
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
2018-09-24
dc.type.none.fl_str_mv research article
http://purl.org/coar/resource_type/c_2df8fbb1
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10486/685952
https://dx.doi.org/10.3389/fnmol.2018.00347
url http://hdl.handle.net/10486/685952
https://dx.doi.org/10.3389/fnmol.2018.00347
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
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
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Frontiers Media S.A.
publisher.none.fl_str_mv Frontiers Media S.A.
dc.source.none.fl_str_mv reponame:Biblos-e Archivo. Repositorio Institucional de la UAM
instname:Universidad Autónoma de Madrid
instname_str Universidad Autónoma de Madrid
reponame_str Biblos-e Archivo. Repositorio Institucional de la UAM
collection Biblos-e Archivo. Repositorio Institucional de la UAM
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869418174805442560
score 15,301603

Similar Items