[3H]WIN 35,428 Binding Assay

Binding assays were conducted to determine whether mutated hDAT alters the kinetic parameters (B_max or K_d) of [³H]WIN 35,428 binding in intact PC12 cells transfected with WT hDAT or mutants. Cells were washed with sucrose-phosphate buffer (final concentration in mM: 2.1 NaH₂PO₄, 7.3 Na₂HPO₄7H₂O, and 320 sucrose, pH 7.4) and then incubated with one of the six concentrations of [³H]WIN 35,428 (84 Ci/mmol, PerkinElmer, 0.5–30 nM final concentrations) in a final volume of 500 μl on ice for 2 h. In parallel, nonspecific binding at each concentration of [³H]WIN 35,428 (in the presence of 30 μM cocaine, final concentration) was subtracted from total binding to calculate the specific binding. For the competitive inhibition experiment, assays were performed in duplicate in a final volume of 500 μl. Intact cells transfected with WT hDAT or its mutants were incubated in buffer containing 50 μl of [³H]WIN 35,428 (final concentration, 5 nM) and one of seven concentrations of unlabeled substrate DA (1 nM–100 μM), cocaine (1 nM–100 μM), GBR12909 (0.01 nM–1 μM) or ZnCl₂ (1, 10, 100 μM) on ice for 2 h. Assays were terminated by removal of reaction reagents in well and then washed three times with ice-cold assay buffer. Cells were lysed with 1% SDS for an hour. Radioactivity was determined as described above.