FIGURE 7.

Effect of imposed membrane proton gradient 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 nigericin (0.12 μg/mg phospholipid) (○) to impose the artificial membrane proton gradient. The vehicle of nigericin, ethanol (●) was used as control of nigericin treatment. The transport assay was started adding 500 μM of [³H]glutamate together with 20 mM Na-gluconate and 30 mM K-gluconate to proteoliposomes containing 10 mM glutamine (A). In (B), the transport was performed in the absence of external K-gluconate, as control. In the figure, sketch of the experimental set up (proteoliposomes); Nig: nigericin. The transport was measured at the indicated times according to the stop inhibitor method. Data were plotted according to the first-order rate equation. Results are means ± SD from four independent experiments.