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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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Autores principales: Benito-Muñoz, Cristina, Perona, Almudena, Abia, David, dos Santos, Helena G., Núñez, Enrique, Aragón, Carmen, López-Corcuera, Beatriz
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166138/
https://www.ncbi.nlm.nih.gov/pubmed/30319354
http://dx.doi.org/10.3389/fnmol.2018.00347
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author Benito-Muñoz, Cristina
Perona, Almudena
Abia, David
dos Santos, Helena G.
Núñez, Enrique
Aragón, Carmen
López-Corcuera, Beatriz
author_facet Benito-Muñoz, Cristina
Perona, Almudena
Abia, David
dos Santos, Helena G.
Núñez, Enrique
Aragón, Carmen
López-Corcuera, Beatriz
author_sort Benito-Muñoz, Cristina
collection PubMed
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.
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spelling pubmed-61661382018-10-12 Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport Benito-Muñoz, Cristina Perona, Almudena Abia, David dos Santos, Helena G. Núñez, Enrique Aragón, Carmen López-Corcuera, Beatriz Front Mol Neurosci Neuroscience 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. Frontiers Media S.A. 2018-09-24 /pmc/articles/PMC6166138/ /pubmed/30319354 http://dx.doi.org/10.3389/fnmol.2018.00347 Text en Copyright © 2018 Benito-Muñoz, Perona, Abia, dos Santos, Núñez, Aragón and López-Corcuera. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Neuroscience
Benito-Muñoz, Cristina
Perona, Almudena
Abia, David
dos Santos, Helena G.
Núñez, Enrique
Aragón, Carmen
López-Corcuera, Beatriz
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
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_short 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
topic Neuroscience
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166138/
https://www.ncbi.nlm.nih.gov/pubmed/30319354
http://dx.doi.org/10.3389/fnmol.2018.00347
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