Functional characterization of C-terminal fusion mGAT1XFP constructs. (A) 20-min [³H]GABA uptake from N2a cells transfected with 100 ng/well of mGAT1 wild-type plasmid, an equimolar amount of the fluorescently tagged mGAT1 plasmids, or blank pcDNA3.1(+) vector. 100% wild-type [³H]GABA uptake is 21.4 ± 1.8 fmol/µg/min. Results represent the mean ± SEM of 6–18 transfections for each construct. *, significant difference compared with wild-type; P ≤ 0.05 (ANOVA with Tukey's post-hoc test). (B) Surface biotinylation experiments comparing the plasma membrane partitioning of mGAT1XFP constructs that had displayed wild-type–like [³H]GABA uptake and one that presents an uptake deficit. Representative bands of the biotinylated mGAT1 surface fractions are displayed above the graph. Bands are arranged in the image to correspond to the appropriate column in the graph. Results represent the mean ± SEM of five to eight transfections for each construct. *, significant difference compared with wild-type; P ≤ 0.05 (one-way ANOVA with Tukey's post-hoc test). (C–E) GABA concentration dependence of six selected constructs whose uptake was closest to wild type in A. Each panel presents three groups of experiments in which the constructs were tested in pairs versus wild-type mGAT1 (100 ng cDNA transfected per 12-well plate well). The nonspecific uptake was determined from wells transfected with empty pcDNA3.1(+) vector and subtracted from the test samples, but is displayed for reference in each plot. No significant difference was found between any of these fluorescent mGAT1 constructs and wild-type mGAT1 for all concentrations, as determined by one-way ANOVA with Tukey's post hoc test (P ≤ 0.05). Data represent the mean uptake for each concentration ± SEM for six to nine transfections.