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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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Voltage jump–induced current relaxations in EAAT4 and EAAC1. (A) Top trace, voltage jump protocol used to measure glutamate-dependent currents shown in the bottom trace, the protocol starts at −100 mV and ends at 60 mV (EAAT4, left), and −90 to 60 mV (EAAC1, right); bottom trace, a typical signal obtained through subtraction of current traces recorded with glutamate from the control traces without glutamate for EAAT4 (left) and EAAC1 (right). Data were recorded at forward transport conditions (140 mM KSCN intracellular). (B) Voltage dependence of maximum currents relative to the current at 0 mV in the presence of 140 mM SCN− outside the membrane (black, closed triangles) determined with a voltage ramp protocol. Traces below the 0 line were performed with SCN− substituting Cl− in the pipette solution. All of these traces were recorded with a voltage jump protocol shown in A. Blue traces indicate measurements with 140 mM potassium in the pipette, transient (squares) and steady-state currents (circles) were plotted separately. Orange traces were recorded under homoexchange conditions with 140 mM sodium and 10 mM glutamate inside the cell; circles correspond to transient currents, squares to steady-state current, respectively. Magenta traces represent currents measured with oocyte ringer and internal solution similar to conditions inside oocytes (see main text). (C) Steady-state current–voltage relationship of the coupled transport current recorded in the absence of permeant ions (MeS susbtitution) with a voltage ramp protocol. The glutamate concentration was 100 μM.
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fig4: Voltage jump–induced current relaxations in EAAT4 and EAAC1. (A) Top trace, voltage jump protocol used to measure glutamate-dependent currents shown in the bottom trace, the protocol starts at −100 mV and ends at 60 mV (EAAT4, left), and −90 to 60 mV (EAAC1, right); bottom trace, a typical signal obtained through subtraction of current traces recorded with glutamate from the control traces without glutamate for EAAT4 (left) and EAAC1 (right). Data were recorded at forward transport conditions (140 mM KSCN intracellular). (B) Voltage dependence of maximum currents relative to the current at 0 mV in the presence of 140 mM SCN− outside the membrane (black, closed triangles) determined with a voltage ramp protocol. Traces below the 0 line were performed with SCN− substituting Cl− in the pipette solution. All of these traces were recorded with a voltage jump protocol shown in A. Blue traces indicate measurements with 140 mM potassium in the pipette, transient (squares) and steady-state currents (circles) were plotted separately. Orange traces were recorded under homoexchange conditions with 140 mM sodium and 10 mM glutamate inside the cell; circles correspond to transient currents, squares to steady-state current, respectively. Magenta traces represent currents measured with oocyte ringer and internal solution similar to conditions inside oocytes (see main text). (C) Steady-state current–voltage relationship of the coupled transport current recorded in the absence of permeant ions (MeS susbtitution) with a voltage ramp protocol. The glutamate concentration was 100 μM.

Mentions: Although glutamate binding in step 1 was assumed to be electroneutral, this step is associated with an apparent valence of +0.8, due to the rapid electrogenic Na+ binding steps preceding and following glutamate binding. Steps 2, 3, and 4 were assumed to be electrogenic with an apparent valence, zQ, of 0.7 and +1.5, and −1.0, respectively (see Scheme 1). These apparent valencies were selected such that the overall charge moved during one transport cycle was +2.0, according to the stoichiometry determined for EAATs 2 and 3 of 3 Na+, 1 H+, and 1 Glu− cotransported for each K+ counter-transported (Zerangue and Kavanaugh, 1996; Levy et al., 1998). Based on our data, there was no indication that the EAAT4 stoichiometry is different from that of the subtypes 2 and 3. The values of the apparent valencies given above result in simulations that fitted the experimental data best (see Fig. 10). For example, when zQ for kr deviated strongly from −1.0, the current voltage relationship of the steady-state anion and transport currents under forward transport conditions (Fig. 3 B, blue circles; Fig. 3 C) could not be well described. The value of the apparent valencies that is the least defined is that of +1.5 for step 3. Variation of this value from +0.5 to +2 did not result in a significant change in the simulations. This is, of course, expected, since k−2 is the fastest reaction step in this cycle, and, thus, far from being rate limiting for the steady-state current or any of the presteady-state phases observed.


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)

Voltage jump–induced current relaxations in EAAT4 and EAAC1. (A) Top trace, voltage jump protocol used to measure glutamate-dependent currents shown in the bottom trace, the protocol starts at −100 mV and ends at 60 mV (EAAT4, left), and −90 to 60 mV (EAAC1, right); bottom trace, a typical signal obtained through subtraction of current traces recorded with glutamate from the control traces without glutamate for EAAT4 (left) and EAAC1 (right). Data were recorded at forward transport conditions (140 mM KSCN intracellular). (B) Voltage dependence of maximum currents relative to the current at 0 mV in the presence of 140 mM SCN− outside the membrane (black, closed triangles) determined with a voltage ramp protocol. Traces below the 0 line were performed with SCN− substituting Cl− in the pipette solution. All of these traces were recorded with a voltage jump protocol shown in A. Blue traces indicate measurements with 140 mM potassium in the pipette, transient (squares) and steady-state currents (circles) were plotted separately. Orange traces were recorded under homoexchange conditions with 140 mM sodium and 10 mM glutamate inside the cell; circles correspond to transient currents, squares to steady-state current, respectively. Magenta traces represent currents measured with oocyte ringer and internal solution similar to conditions inside oocytes (see main text). (C) Steady-state current–voltage relationship of the coupled transport current recorded in the absence of permeant ions (MeS susbtitution) with a voltage ramp protocol. The glutamate concentration was 100 μM.
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fig4: Voltage jump–induced current relaxations in EAAT4 and EAAC1. (A) Top trace, voltage jump protocol used to measure glutamate-dependent currents shown in the bottom trace, the protocol starts at −100 mV and ends at 60 mV (EAAT4, left), and −90 to 60 mV (EAAC1, right); bottom trace, a typical signal obtained through subtraction of current traces recorded with glutamate from the control traces without glutamate for EAAT4 (left) and EAAC1 (right). Data were recorded at forward transport conditions (140 mM KSCN intracellular). (B) Voltage dependence of maximum currents relative to the current at 0 mV in the presence of 140 mM SCN− outside the membrane (black, closed triangles) determined with a voltage ramp protocol. Traces below the 0 line were performed with SCN− substituting Cl− in the pipette solution. All of these traces were recorded with a voltage jump protocol shown in A. Blue traces indicate measurements with 140 mM potassium in the pipette, transient (squares) and steady-state currents (circles) were plotted separately. Orange traces were recorded under homoexchange conditions with 140 mM sodium and 10 mM glutamate inside the cell; circles correspond to transient currents, squares to steady-state current, respectively. Magenta traces represent currents measured with oocyte ringer and internal solution similar to conditions inside oocytes (see main text). (C) Steady-state current–voltage relationship of the coupled transport current recorded in the absence of permeant ions (MeS susbtitution) with a voltage ramp protocol. The glutamate concentration was 100 μM.
Mentions: Although glutamate binding in step 1 was assumed to be electroneutral, this step is associated with an apparent valence of +0.8, due to the rapid electrogenic Na+ binding steps preceding and following glutamate binding. Steps 2, 3, and 4 were assumed to be electrogenic with an apparent valence, zQ, of 0.7 and +1.5, and −1.0, respectively (see Scheme 1). These apparent valencies were selected such that the overall charge moved during one transport cycle was +2.0, according to the stoichiometry determined for EAATs 2 and 3 of 3 Na+, 1 H+, and 1 Glu− cotransported for each K+ counter-transported (Zerangue and Kavanaugh, 1996; Levy et al., 1998). Based on our data, there was no indication that the EAAT4 stoichiometry is different from that of the subtypes 2 and 3. The values of the apparent valencies given above result in simulations that fitted the experimental data best (see Fig. 10). For example, when zQ for kr deviated strongly from −1.0, the current voltage relationship of the steady-state anion and transport currents under forward transport conditions (Fig. 3 B, blue circles; Fig. 3 C) could not be well described. The value of the apparent valencies that is the least defined is that of +1.5 for step 3. Variation of this value from +0.5 to +2 did not result in a significant change in the simulations. This is, of course, expected, since k−2 is the fastest reaction step in this cycle, and, thus, far from being rate limiting for the steady-state current or any of the presteady-state phases observed.

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