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Correspondence to Christof Grewer: cgrewer{at}med.miami.edu
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, Vm, of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a 
of 
15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<–40 mV) and activation at positive Vm (>0 mV). A similar inhibitory effect at Vm < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-Vm curve. Jumping the glutamate concentration to 100 µM generated biphasic, saturable transient transport and anion currents (Km 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.
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