Fluorescence-tagged Monoamine Transporter Assay

Fluorescence tags do not introduce any functional deficit to monoamine transporters (24 (link)). Uptake experiments were performed as described previously (25 (link)). In brief, for the determination of nonspecific uptake by SERT, DAT, and NET, we used 10 μm paroxetine, mazindole, or nisoxetine, respectively, and 0.1–60 μm [³H]dopamine or [³H]5HT was added for 1 min as indicated. For ^TacSERT, transfected cells were plated in 96-well plates (ViewPlate; PerkinElmer Life Science). Prior to the experiment, the cells were washed once in uptake buffer (25 mm HEPES, 120 mm NaCl, 5 mm KCl, 1.2 mm CaCl₂, and 1.2 mm MgSO₄ supplemented with 5 mm d-glucose, 0.1 mm ascorbic acid, and 0.1 mm pargyline) and equilibrated in uptake buffer for 30 min before starting the assay. Nonlabeled 5HT was added at increasing concentrations followed by 30–50 nm [³H]5HT to a final volume of 150 μl. After incubation for 3 min at room temperature, cells were washed twice in ice-cold uptake buffer. Scintillation fluid (0.15 ml) was added, and the radioactivity was measured in a Wallac microplate liquid scintillation counter (PerkinElmer Life Sciences).
Substrate efflux assays were performed as described previously (26 (link), 27 (link)). In brief, culture medium was removed from transiently transfected CAD or HEK293 cells (4·10⁵ cells per well grown on coverslips in 96-well plates), and the cells were preincubated with 0.4 μm [³H]5HT or with 0.1 μm 1-[³H]methyl-4-phenylpyridinium (MPP⁺) for 20 min at 37 °C in a final volume of 0.1 ml of Krebs/HEPES buffer per well. The coverslips were transferred into chambers, and excess radioactivity was subsequently washed out with buffer at 25 °C for 45 min at a perfusion rate of 0.7 ml/min. Once stable efflux of radioactivity was achieved, following the initial wash, 2-min fractions were collected, and samples were counted in a β-counter.