FIGURE 4.

Effect of imposed membrane potential on glutamate transport by hASCT2 reconstituted in proteoliposomes. Purified hASCT2 was reconstituted in proteoliposomes as described in materials and methods. After sephadex-G75 chromatography, proteoliposomes were treated with valinomycin (0.75 μg/mg phospholipid) to impose the artificial membrane potential. The vehicle of valinomycin, ethanol (EtOH) was used as control of valinomycin treatment. The transport assay was started adding 500 μM of [³H]glutamate together with 50 mM Na-gluconate to proteoliposomes containing 10 mM glutamine (A) or glutamate (B) and 50 mM K-gluconate. The transport was measured in 60 min according to the stop inhibitor method. The transport rate was expressed as nmol/mg in 60 min. In (A,B) sketch of the experimental set up (proteoliposomes); Val: valinomycin. In (C), data from (A,B) were used to calculate the fold of activation valinomycin/ethanol. Results are means ± SD from four independent experiments. *Significantly different from the control sample (EtOH) as estimated by Student’s t-test (P < 0.05).