[3H]spiperone binding assay

FlpIn CHO cells (Invitrogen) stably expressing WT or mutant SNAP-D2s cells were cultured before the preparation of cell membrane as described before (Klein Herenbrink et al., 2019). All stable cell lines were confirmed to be mycoplasma free. For saturating binding assays cell membranes (Mutant or WT SNAP-D_2s-FlpIn CHO, 2.5 µg) were incubated with varying concentrations of [³H]spiperone and 10 µM haloperidol as a non-specific control, in binding buffer (20 mM HEPES, 100 mM NaCl, 6 mM MgCl₂, 1 mM EGTA, and 1 mM EDTA, pH 7.4) to a final volume of 200 μL and were incubated at 37°C for 3 hr. For competition binding assays, cell membranes (SNAP-D_2s-FlpIn CHO, 2.5 µg) were incubated with varying concentrations of test compound in binding buffer containing 0.2 nM of [³H]spiperone to a final volume of 200 µL and were incubated at 37°C for 3 hr. Binding was terminated by fast-flow filtration using a Uniplate 96-well harvester (PerkinElmer) followed by five washes with ice-cold 0.9% NaCl. Bound radioactivity was measured in a MicroBeta2 LumiJET MicroBeta counter (PerkinElmer). Data were collected from at least three separate experiments performed in triplicate and analysed using non-linear regression (Prism 7, Graphpad software). For radioligand saturation binding data, the following equation was globally fitted to nonspecific and total binding data:

where Y is radioligand binding, B_max is the total receptor density, [A] is the free radioligand concentration, K_A is the equilibrium dissociation constant of the radioligand, and NS is the fraction of nonspecific radioligand binding. The B_max of the SNAP-tagged D2SRs we as follows; WT = 7.95 ± 1.63 pmol.mg⁻¹, 6.39 ± 1.04 pmol.mg⁻¹, 4.37 ± 0.92 pmol.mg⁻¹, 2.61 ± 0.50 pmol.mg⁻¹.

For competition binding assays, the concentration of ligand that inhibited half of the [³H]spiperone binding (IC₅₀) was determined by fitting the data to the following equation:

where Y denotes the percentage specific binding, Top and Bottom denote the maximal and minimal asymptotes, respectively, IC₅₀ denotes the X-value when the response is midway between Bottom and Top, and nH denotes the Hill slope factor. IC₅₀ values obtained from the inhibition curves were converted to K_i values using the Cheng and Prusoff equation. No statistical methods were used to predetermine sample size.