Fig. 2.

Cellular expression and functional characterization of wild-type and mutant SERTs. a, the cellular expression of wild-type or mutant SERTs was examined by confocal laser scanning microscopy. Representative images of HEK293 cells (10⁵ cells/16-mm glass coverslip) stably expressing YFP-tagged transporters indicate that the expression pattern of mutated proteins are similar to those shown by the wild-type SERT. b, for saturation uptake experiments, HEK293 cells stably expressing wild-type or mutant SERTs were seeded onto 48-well plates (0.5 × 10⁵ cells/well) 24 h before experiments. Cells were incubated with [³H]5-HT with or without 10 μM paroxetine to determine nonspecific uptake. The assay was conducted in duplicates and reaction stopped after 1 min with ice-cold Krebs-HEPES buffer. Data, plotted according to the hyperbolic model, are shown as means of a representative experiment carried out in duplicate. c, imipramine binding to SERT^WT and SERT^Y95F: the incubation was performed in a final volume of 250 μl for 15 min at 27°C. Nonspecific binding was determined in the presence of 3 μM paroxetine and was ≤10% at the K_D concentration of SERT^WT. Data are from a single experiment carried out in duplicate, which was repeated three times. Average B_max values were 3.6 ± 0.1 and 4.0 ± 0.3 pmol/mg for SERT^WT and SERT^Y95F, respectively. The B_max of the mutant is likely to be underestimated: because of the low affinity of [³H]imipramine, some bound ligand dissociates during the separation artifact. This conjecture was verified by immunoblotting (insert), which shows substantially higher levels of SERT^Y95F than of SERT^WT. The immunoblot revealed two bands: the top band corresponds to the mature, fully glycosylated form, and the bottom form corresponds to the core-glycosylated (endoplasmic reticulum resident) form. d, inhibition of [³H]5-HT uptake by imipramine was determined in HEK293 stably expressing wild-type and mutant SERTs. The cells were incubated with 0.1 nM [³H]5-HT and increasing concentrations of imipramine, in the presence or absence of 10 μM paroxetine (to determine nonspecific uptake). The data (n = 3) were analyzed as described under Materials and Methods and plotted according to the sigmoidal model.