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The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism
Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-μs time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion cur...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
2005
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2266596/ https://www.ncbi.nlm.nih.gov/pubmed/16316976 http://dx.doi.org/10.1085/jgp.200509365 |
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author | Mim, Carsten Balani, Poonam Rauen, Thomas Grewer, Christof |
author_facet | Mim, Carsten Balani, Poonam Rauen, Thomas Grewer, Christof |
author_sort | Mim, Carsten |
collection | PubMed |
description | Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-μs time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na(+) with high affinities of 0.6 μM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na(+). The steady-state cycle time of EAAT4 was estimated to be >300 ms (at −90 mV). Applying step changes to the transmembrane potential, V (m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a τ of ∼15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<−40 mV) and activation at positive V (m) (>0 mV). A similar inhibitory effect at V (m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V (m) curve. Jumping the glutamate concentration to 100 μM generated biphasic, saturable transient transport and anion currents (K (m) ∼ 5 μM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na(+) binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1–3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes. |
format | Text |
id | pubmed-2266596 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2005 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22665962008-03-21 The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism Mim, Carsten Balani, Poonam Rauen, Thomas Grewer, Christof J Gen Physiol Article Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-μs time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na(+) with high affinities of 0.6 μM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na(+). The steady-state cycle time of EAAT4 was estimated to be >300 ms (at −90 mV). Applying step changes to the transmembrane potential, V (m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a τ of ∼15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<−40 mV) and activation at positive V (m) (>0 mV). A similar inhibitory effect at V (m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V (m) curve. Jumping the glutamate concentration to 100 μM generated biphasic, saturable transient transport and anion currents (K (m) ∼ 5 μM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na(+) binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1–3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes. The Rockefeller University Press 2005-12 /pmc/articles/PMC2266596/ /pubmed/16316976 http://dx.doi.org/10.1085/jgp.200509365 Text en Copyright © 2005, The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Article Mim, Carsten Balani, Poonam Rauen, Thomas Grewer, Christof The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism |
title | The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism |
title_full | The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism |
title_fullStr | The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism |
title_full_unstemmed | The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism |
title_short | The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism |
title_sort | glutamate transporter subtypes eaat4 and eaats 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2266596/ https://www.ncbi.nlm.nih.gov/pubmed/16316976 http://dx.doi.org/10.1085/jgp.200509365 |
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