2.4.3. Transporter binding assays
Membrane preparations expressing the transporters hDAT and hSERT were incubated with radiolabelled selective ligands at concentrations equal or close to Kd, and ligand displacement by the tested drugs at different concentrations was measured. The drugs were diluted in binding buffer (120 mM NaCl, 3 mM KCl, 2 mM MgCl2, 10 µM ZnCl2 and 20 mM Tris pH 7,4 for hDAT, and 120 mM NaCl, 3 mM KCl, 2 mM MgCl2, 1 mM EDTA and 20 mM Tris pH 7,4 for hSERT) and tested at increasing concentrations (ranged from 0,1 nM – 300 µM) in duplicate. The binding reactions were performed in tubes containing 25 µl of the radioligand: [3H]WIN35,428 (hDAT assay, final concentration 10 nM) or [3H]imipramine (hSERT assay, final concentration 3 nM) diluted in the corresponding reaction buffer, 5 µg of membranes and 100 µl of the tested drug dilution. Non-specific binding was determined in the presence of cocaine 100 µM (for hDAT radioligand) and paroxetine 3 µM (for hSERT radioligand). Incubation was performed for 1 hour at 20 ºC. The binding reactions were terminated by rapid filtration of the membranes through GF/C glass microfiber filters pre-soaked with 0,5 % polyethyleneimine and rapid washing with ice-cold wash buffer (120 nM NaCl, 2 mM MgCl2, 10 mM Tris and 100 µM ZnCl2 for hDAT, and 120 nM NaCl, 2 mM MgCl2 and 10 mM Tris, for hSERT). Afterwards, scintillation cocktail was added to the vials containing the filters, and the trapped radioactivity was quantified by liquid scintillation counting. Specific binding of each compound to the transporter was defined as the difference between total binding (binding buffer alone) and non-specific binding. All determinations were performed per triplicate. All experiments were performed three times (N=3).