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GLT-1 Transport Stoichiometry Is Constant at Low and High Glutamate Concentrations when Chloride Is Substituted by Gluconate

Glutamate is the major excitatory neurotransmitter, but prolonged exposure even at micromolar concentrations causes neuronal death. Extracellular glutamate is maintained at nanomolar level by glutamate transporters, which, however, may reverse transport and release glutamate. If and when the reverse...

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Autores principales: Kabakov, Anatoli Y., Rosenberg, Paul A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547712/
https://www.ncbi.nlm.nih.gov/pubmed/26301411
http://dx.doi.org/10.1371/journal.pone.0136111
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author Kabakov, Anatoli Y.
Rosenberg, Paul A.
author_facet Kabakov, Anatoli Y.
Rosenberg, Paul A.
author_sort Kabakov, Anatoli Y.
collection PubMed
description Glutamate is the major excitatory neurotransmitter, but prolonged exposure even at micromolar concentrations causes neuronal death. Extracellular glutamate is maintained at nanomolar level by glutamate transporters, which, however, may reverse transport and release glutamate. If and when the reverse occurs depends on glutamate transport stoichiometry (GTS). Previously we found that in the presence of chloride, the coupled GLT-1 glutamate transporter current and its relationship to radiolabeled glutamate flux significantly decreased when extracellular glutamate concentration increased above 0.2 mM, which implies a change in GTS. Such high concentrations are feasible near GLT-1 expressed close to synaptic release site during excitatory neurotransmission. The aim of this study was to determine GLT-1 GTS at both low (19–75 μM) and high (300–1200 μM) glutamate concentration ranges. GTS experiments were conducted in the absence of chloride to avoid contributions by the GLT-1 uncoupled chloride conductance. Mathematical analysis of the transporter thermodynamic equilibrium allowed us to derive equations revealing the number of a particular type of ion transported per elementary charge based on the measurements of the transporter reversal potential. We found that GLT-1a expressed in COS-7 cells co-transports 1.5 Na(+), 0.5 Glu(-), 0.5 H(+) and counter-transports 0.6 K(+) per elementary charge in both glutamate concentration ranges, and at both 37°C and 26°C temperatures. The thermodynamic parameter Q (10) = 2.4 for GLT-1 turnover rate of 19 s(-1) (37°C, -50 mV) remained constant in the 10 μM–10 mM glutamate concentration range. Importantly, the previously reported decrease in the current/flux ratio at high glutamate concentration was not seen in the absence of chloride in both COS-7 cells and cultured rat neurons. Therefore, only in the absence of chloride, GLT-1 GTS remains constant at all glutamate concentrations. Possible explanations for why apparent GTS might vary in the presence of chloride are discussed.
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spelling pubmed-45477122015-09-01 GLT-1 Transport Stoichiometry Is Constant at Low and High Glutamate Concentrations when Chloride Is Substituted by Gluconate Kabakov, Anatoli Y. Rosenberg, Paul A. PLoS One Research Article Glutamate is the major excitatory neurotransmitter, but prolonged exposure even at micromolar concentrations causes neuronal death. Extracellular glutamate is maintained at nanomolar level by glutamate transporters, which, however, may reverse transport and release glutamate. If and when the reverse occurs depends on glutamate transport stoichiometry (GTS). Previously we found that in the presence of chloride, the coupled GLT-1 glutamate transporter current and its relationship to radiolabeled glutamate flux significantly decreased when extracellular glutamate concentration increased above 0.2 mM, which implies a change in GTS. Such high concentrations are feasible near GLT-1 expressed close to synaptic release site during excitatory neurotransmission. The aim of this study was to determine GLT-1 GTS at both low (19–75 μM) and high (300–1200 μM) glutamate concentration ranges. GTS experiments were conducted in the absence of chloride to avoid contributions by the GLT-1 uncoupled chloride conductance. Mathematical analysis of the transporter thermodynamic equilibrium allowed us to derive equations revealing the number of a particular type of ion transported per elementary charge based on the measurements of the transporter reversal potential. We found that GLT-1a expressed in COS-7 cells co-transports 1.5 Na(+), 0.5 Glu(-), 0.5 H(+) and counter-transports 0.6 K(+) per elementary charge in both glutamate concentration ranges, and at both 37°C and 26°C temperatures. The thermodynamic parameter Q (10) = 2.4 for GLT-1 turnover rate of 19 s(-1) (37°C, -50 mV) remained constant in the 10 μM–10 mM glutamate concentration range. Importantly, the previously reported decrease in the current/flux ratio at high glutamate concentration was not seen in the absence of chloride in both COS-7 cells and cultured rat neurons. Therefore, only in the absence of chloride, GLT-1 GTS remains constant at all glutamate concentrations. Possible explanations for why apparent GTS might vary in the presence of chloride are discussed. Public Library of Science 2015-08-24 /pmc/articles/PMC4547712/ /pubmed/26301411 http://dx.doi.org/10.1371/journal.pone.0136111 Text en © 2015 Kabakov, Rosenberg http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Kabakov, Anatoli Y.
Rosenberg, Paul A.
GLT-1 Transport Stoichiometry Is Constant at Low and High Glutamate Concentrations when Chloride Is Substituted by Gluconate
title GLT-1 Transport Stoichiometry Is Constant at Low and High Glutamate Concentrations when Chloride Is Substituted by Gluconate
title_full GLT-1 Transport Stoichiometry Is Constant at Low and High Glutamate Concentrations when Chloride Is Substituted by Gluconate
title_fullStr GLT-1 Transport Stoichiometry Is Constant at Low and High Glutamate Concentrations when Chloride Is Substituted by Gluconate
title_full_unstemmed GLT-1 Transport Stoichiometry Is Constant at Low and High Glutamate Concentrations when Chloride Is Substituted by Gluconate
title_short GLT-1 Transport Stoichiometry Is Constant at Low and High Glutamate Concentrations when Chloride Is Substituted by Gluconate
title_sort glt-1 transport stoichiometry is constant at low and high glutamate concentrations when chloride is substituted by gluconate
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547712/
https://www.ncbi.nlm.nih.gov/pubmed/26301411
http://dx.doi.org/10.1371/journal.pone.0136111
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