Ligand Association and Dissociation Radioligand Binding Assays

Binding assays were performed using HEK293 T membrane preparations transiently expressing DRD2 (D₂ long receptor) and different mutants at room temperature. Radioligand dissociation and association assays were performed in parallel utilizing the same concentrations of radioligand, membrane preparations, and binding buffer (50 mM Tris, 10 mM MgCl₂, 0.1 mM EDTA, 0.1% BSA, pH 7.4). All assays utilized at least 2 concentrations of radioligand ([0.5–1.0 nM [³H]-N-methylspiperone; 0.5–2.0 nM [³H]-nemonapride). For dissociation assays, membranes were incubated with radioligand for at least 2 hr at room temperature before the addition of 10 μL of 10 μM excess cold ligand to the 200 μL membrane suspension at designated time points. For association experiments, 100 μL of radioligand was added to 100 μL membrane suspensions at designated time points. Time points spanned 1 minutes to 7 hr, depending on experimental conditions and radioligand. For the determination of k_on and k_off for unlabeled risperidone or aripiprazole, membranes of either wild type or mutants were incubated with [³H]-methylspiperone and several concentrations of risperidone or aripiprazole. Non-specific binding was determined by addition of 10 μM nemonapride. Immediately at time = 0 min, plates were harvested by vacuum filtration onto 0.3% polyethyleneimine pre-soaked 96-well filter mats (Perkin Elmer) using a 96-well Filtermate harvester, followed by three washes of cold wash buffer (50 mM Tris pH 7.4). Scintillation cocktail (Meltilex, Perkin Elmer) was melted onto dried filters and radioactivity was counted using a Wallac Trilux MicroBeta counter (PerkinElmer). Data were analyzed using “Dissociation-One phase exponential decay” or “Association kinetics-Two or more concentrations of hot radioligand” in Graphpad Prism 5.0. The previously determined [³H]-N-methylspiperone k_on and k_off rates of DRD2 or mutants was used to estimate the k_on and k_off rates of risperidone and aripiprazole using the “Kinetics of competitive binding” equation in Graphpad Prism 5.0 as put forth by Motulsky and Mahan³⁹.