Radioligand binding assays

Membrane proteins prepared from Sf9 cells expressing dopamine D₂ (25 μg) or dopamine D₃ receptors (10 μg) were incubated with a range of concentrations of [³H]spiperone in buffer 2 (20 mM HEPES, 6 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 0.1% BSA, pH 7.4) supplemented, where appropriate, with 100 mM NaCl or 100 mM N-methyl-D-glucamine (NMDG). Reactions were performed, in triplicate, in 4 ml LP4 test tubes (1 ml final volume) and were initiated by addition of membrane proteins. Non-specific binding was determined in the presence of (+)-butaclamol (3 μM). Reactions were incubated for 3 hours at 25°C and were terminated by rapid filtration through Whatman glass microfibre GF/C filters using a Brandel cell harvester. After four 3 ml washes with PBS (4°C) filter discs were transferred to scintillation vials and soaked in 2ml Ultima Gold^™ XR scintillation fluid (Perkin Elmer) for at least 6 hours prior to their radioactivity being determined by liquid scintillation spectrometry. Specific binding was calculated by subtraction of non-specific binding and free radioligand concentration, corrected for ligand depletion calculated. Data were analysed using Prism (Graphpad) and were fitted to hyperbolic equations describing a one-binding site model.

Membrane protein prepared from U-2 OS cells (2 μg) or Sf9 cells (25 μg [D₂] or 10 μg [D₃]) expressing wild-type or mutant receptors was incubated with a fixed concentration of [³H]spiperone (0.25 nM [D₂] or 1 nM [D₃]) and a range of concentrations of competing ligand in buffer 2 (20 mM HEPES, 6 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 0.1% BSA, pH 7.4) supplemented with dithiothreitol (0.1 nM) and, where appropriate, 100 mM NaCl or 100 mM NMDG. Non-specific binding was defined using (+)-butaclamol (3 μM) in place of the competing ligand. Reactions were performed, in triplicate, in 4 ml LP4 test tubes (1 ml final volume) [Sf9] or deep-well 96-well plates (400μl final volume) [U-2 OS]. Non-specific binding was determined in the presence of (+)-butaclamol (3 μM) and total binding determined in the absence of competing ligand. Reactions were initiated, incubated, terminated and radioactivity measured as described under ‘Radioligand saturation binding assays’. Data are presented as percentage of total binding, after subtraction of non-specific binding. Data were analysed using Prism (Graphpad) and were fitted to sigmoidal equations describing a one-binding site model. Where % inhibition was <100%, parameters were calculated by data extrapolation.

Statistical significance of differences between binding parameters were calculated by one-way ANOVA followed by Tukey’s post-hoc test, with a value of p<0.05 considered significant. Before statistical analysis IC₅₀ and, K_d values were converted to their respective normally distributed negative logarithms (pIC₅₀ and pK_d).