FIGURE 5.
Uptake of [3H]GABA in proteoliposomes. Proteoliposomes were prepared from HeLa cells expressing the indicated GAT-1 mutants as described under “Experimental Procedures” using 10 μl of proteoliposomes containing 15–30 μg of protein, which were diluted into 360 μl of 75 mm NaCl plus 60 mm NaPi, pH 7.4, supplemented with 1 μCi of [3H]GABA and 2.8 μm valinomycin for each triplicate time point (10 min). The “in” medium contained either 120 mm KPi, pH 7.4 (gray bars) or 150 mm KMES, pH 6.0 (black bars). The data are given as percentages of activity of GAT-1-WT at pH 7.4, which ranged from 15.2–28.0 pmol/mg·protein, and are the means ± S.E. of at least four separate experiments.