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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.

Mim C, Balani P, Rauen T, Grewer C - J. Gen. Physiol. (2005)

Bottom Line: 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.The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation.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.

View Article: PubMed Central - PubMed

Affiliation: University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
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-micros 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 microM 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 tau of approximately 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 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) 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.

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Pseudo four-state model used for kinetic modeling of EAAT4 anion currents. The detailed kinetic parameters used for modeling are listed in table 1.
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fig2: Pseudo four-state model used for kinetic modeling of EAAT4 anion currents. The detailed kinetic parameters used for modeling are listed in table 1.

Mentions: We have used a simplified four-step sequential transport model to explain the experimental data. This model includes (a) a reversible glutamate binding reaction to the transporter on the extracellular side, characterized by the rate constants k+1 and k-1. Binding and dissociation of glutamate were assumed to be modulated by the preceding and subsequent Na+ binding steps, which were assumed to be in rapid preequilibrium with respect to glutamate binding. Thus, the overall reaction from T to \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\overline{N_{2}TG}\end{equation*}\end{document} reduces to an apparent one-step process. (b) A reversible glutamate translocation reaction (rate constants kt and k-t). Glutamate translocation was assumed to be reversible for modeling of the exchange mode. In this mode, high intracellular concentrations of glutamate and Na+ were used that saturate their intracellular binding sites. Thus, glutamate and Na+ are prevented to dissociate to the cytoplasm, locking the transporter in states \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\overline{N_{2}TG}\end{equation*}\end{document} and \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\overline{N_{2}T^{\prime}G}\end{equation*}\end{document} (shaded area in Scheme 1). The model also includes two under forward transport conditions quasi-irreversible transport steps. (c) A reaction characterized by k-2 which could be linked to any of the intracellular substrate dissociation steps, such as glutamate dissociation (see Scheme 1) or Na+ dissociation. This step was assumed to be quasi-irreversible because the cytoplasmic Na+ and glutamate concentrations under forward transport conditions should be nominally zero. Therefore, Na+ and glutamate are prevented from associating to the transporter after dissociation has taken place. (d) The K+-driven relocation reaction characterized by the rate constant kr, which might be either the K+ translocation reaction, association with intracellular K+, or dissociation of extracellular K+. This step was also assumed to be quasi-irreversible because the extracellular [K+] was zero, thus preventing reassociation of extracellular K+ with EAAT4 and driving the transporter into state T (Scheme 1).


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 C, Balani P, Rauen T, Grewer C - J. Gen. Physiol. (2005)

Pseudo four-state model used for kinetic modeling of EAAT4 anion currents. The detailed kinetic parameters used for modeling are listed in table 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266596&req=5

fig2: Pseudo four-state model used for kinetic modeling of EAAT4 anion currents. The detailed kinetic parameters used for modeling are listed in table 1.
Mentions: We have used a simplified four-step sequential transport model to explain the experimental data. This model includes (a) a reversible glutamate binding reaction to the transporter on the extracellular side, characterized by the rate constants k+1 and k-1. Binding and dissociation of glutamate were assumed to be modulated by the preceding and subsequent Na+ binding steps, which were assumed to be in rapid preequilibrium with respect to glutamate binding. Thus, the overall reaction from T to \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\overline{N_{2}TG}\end{equation*}\end{document} reduces to an apparent one-step process. (b) A reversible glutamate translocation reaction (rate constants kt and k-t). Glutamate translocation was assumed to be reversible for modeling of the exchange mode. In this mode, high intracellular concentrations of glutamate and Na+ were used that saturate their intracellular binding sites. Thus, glutamate and Na+ are prevented to dissociate to the cytoplasm, locking the transporter in states \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\overline{N_{2}TG}\end{equation*}\end{document} and \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\overline{N_{2}T^{\prime}G}\end{equation*}\end{document} (shaded area in Scheme 1). The model also includes two under forward transport conditions quasi-irreversible transport steps. (c) A reaction characterized by k-2 which could be linked to any of the intracellular substrate dissociation steps, such as glutamate dissociation (see Scheme 1) or Na+ dissociation. This step was assumed to be quasi-irreversible because the cytoplasmic Na+ and glutamate concentrations under forward transport conditions should be nominally zero. Therefore, Na+ and glutamate are prevented from associating to the transporter after dissociation has taken place. (d) The K+-driven relocation reaction characterized by the rate constant kr, which might be either the K+ translocation reaction, association with intracellular K+, or dissociation of extracellular K+. This step was also assumed to be quasi-irreversible because the extracellular [K+] was zero, thus preventing reassociation of extracellular K+ with EAAT4 and driving the transporter into state T (Scheme 1).

Bottom Line: 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.The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation.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.

View Article: PubMed Central - PubMed

Affiliation: University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
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-micros 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 microM 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 tau of approximately 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 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) 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.

Show MeSH
Related in: MedlinePlus