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First published online May 24, 2005
Journal of Experimental Biology 208, 2063-2070 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01595

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Glutamate transporter type 3 attenuates the activation of N-methy-D-aspartate receptors co-expressed in Xenopus oocytes

Zhiyi Zuo^* and Hongyu Fang

Department of Anesthesiology, University of Virginia Health System, Charlottesville, Virginia 22908-0710, USA

View larger version (13K): [in a new window]   Fig. 1. Glutamate dose-response curves of NMDAR (open circle) and EAAT3 (filled circle). Oocytes were injected with 40 ng of NMDAR mRNA or 40 ng of EAAT3 mRNA. Results are means ±S.D. (N=16 for NMDAR and N=12 for EAAT3) of the percentage of the currents induced by various concentrations of glutamate (µmol l-l) in the current induced by 300 µmol l-1 of glutamate (Imax).

View larger version (12K): [in a new window]   Fig. 2. Stopped-flow reduction of NMDAR current in EAAT3+ oocytes. NMDAR was activated continuously (dashed line) by a solution containing 3 µmol l-1 glutamate. Superfusate flow was stopped (solid line) for about 15 s during the activation. (A) Glutamate-induced current was decreased when the flow was stopped in EAAT3+ oocytes, (B) whereas the interruption of the flow had no effect on glutamate-induced current in EAAT3- oocytes. Restarting the flow for 5 s restored the full amplitude of the glutamate-induced current in EAAT3+ oocytes.

View larger version (15K): [in a new window]   Fig. 3. Stopped-flow reduction of NMDAR current in EAAT3+ oocytes requires function of EAAT3. Stopped-flow reduction of NMDAR current (A) was not observed in EAAT3+ oocytes when Na+ was replaced by Li+ in the superfusates (B) or when NMDA was the agonist for NMDAR in the superfusate (C).

View larger version (20K): [in a new window]   Fig. 4. Dose-dependence of the stopped-flow reduction of NMDAR current in EAAT3+ oocytes. Various concentrations (numbers on the right-hand side of the graph, µmol l-1) of glutamate were applied to the oocytes and the current traces were normalized to the trace induced by 100 µmol l-1 glutamate.

View larger version (13K): [in a new window]   Fig. 5. Comparison of glutamate concentrations sensed by NMDAR (solid line) with those in the superfusates in the co-expressing oocytes. The dashed line refers to the situation that when the glutamate concentrations sensed by NMDAR equal to the glutamate concentrations in the superfusates. The sensed glutamate concentrations are calculated based on equation (1). Results are means ±S.D. of sensed glutamate concentrations (N=12).

View larger version (19K): [in a new window]   Fig. 6. Current traces showing that the reduction of glutamate-induced current under continuous flow conditions is established rapidly. The glutamate-induced current was produced repeatedly by 25 s applications of solutions containing various concentrations of glutamate (numbers in the figure, µmol l-1) to oocytes expressing NMDAR only (EAAT3- oocytes in panel A) or co-expression oocytes (EAAT3+ oocytes in panel B).

View larger version (21K): [in a new window]   Fig. 7. Reduction of NMDAR current in EAAT3+ oocytes under continuous flow conditions. (A) Glutamate (µmol l-1) dose-response curves of EAAT3. Oocytes were injected with 40 ng of EAAT3 mRNA (filled circle) or 40 ng of NMDAR mRNA and 40 ng of EAAT3 mRNA (co-expression, open circle). In co-expressing oocytes, glutamate was in a glycine-free solution containing 5 mmol l-1 Mg2+ to inhibit the NMDAR activation. Results are means ±S.D. (N=12 for EAAT3 single expressing oocytes and N=9 for co-expressing oocytes) of the percentage of the currents induced by various concentrations of glutamate in the maximal EAAT3 current induced by 300 µmol l-1 of glutamate (Imax). (B) NMDA (µmol l-1) dose-response curves of NMDAR. Oocytes were injected with 40 ng of NMDAR mRNA (filled circle) or 40 ng of NMDAR mRNA and 40 ng of EAAT3 mRNA (open circle). In co-expressing oocytes, no EAAT inhibitors were included in the superfusates. Results are means ±S.D. (N=6 for both NMDAR single expressing oocytes and co-expressing oocytes) of the percentage of the currents induced by various concentrations of glutamate in the maximal NMDAR current induced by 1 mmol of NMDA (Imax). (C) Dose-response curves of glutamate-induced current. Oocytes were injected with 40 ng of NMDAR mRNA (filled circle) or 40 ng of NMDAR mRNA and 40 ng of EAAT3 mRNA (open circle and open triangle) or 10 ng of NMDAR mRNA and 40 ng of EAAT3 mRNA (filled triangle). EAAT3 activity was inhibited by replacing Na+ with Li+ in the solution and the data are presented as open triangle in the graph. Results are means ±S.D. (N=16 for filled circle, N=15 for open circle, N=10 for filled triangle and N=6 for the open triangle) of the percentage of the currents induced by various concentrations of glutamate in the maximal current induced by 300 µmol l-1 of glutamate (Imax). (D) Relationship between the EC50 shift of the dose-response curves of glutamate-induced current and the percentage of EAAT3 current in the total glutamate-induced current in the co-expressing oocytes. EAAT3 current was isolated from NMDAR current by using a glycine-free solution containing 5 mmol l-1 Mg2+ to inhibit the NMDAR activation/current. Each data point represents data from one oocyte. A linear regression yielded an r=0.607 (P<0.05).