4.2. D2 and D3 Dopamine Receptor Competitive Radioligand Binding Assays

For these competitive binding studies, transfected HEK293 cell homogenates were suspended in homogenization buffer and incubated with radioligand [¹²⁵I]IABN, in the presence or absence of inhibitor at 37 °C for 60 min (total volume = 150 μL), as previously described [49]. The final radioligand concentration was approximately equal to the K_d value for the binding of the radioligand. Nonspecific binding was defined as the binding of the radioligand in the presence of 25 µM (+)-butaclamol. For each competition curve, triplicates were performed using two concentrations of inhibitor per decade over five orders of magnitude. Binding was terminated by the addition of cold wash buffer (10 mM Tris–HCl/150 mM NaCl, pH = 7.5) and filtration over a glass-fiber filter (Pall A/B filters, #66198). A Packard Cobra Gamma Counter (PerkinElmer, Waltham, MA, USA) was used to measure the radioactivity of [¹²⁵I]IABN.

The competition curves were modeled for a single binding site using

where Bs is the amount of ligand bound to receptor and Bo is the amount of ligand bound to receptor in the absence of competitive inhibitor. L is the concentration of the competitive inhibitor. The IC₅₀ value is the concentration of competitive inhibitor that inhibits 50% of the total specific binding. IC₅₀ values were determined using non-linear regression analysis with TableCurve 2D v 5.01 (Jandel, SYSTAT, Systat Software, Inc., San Jose, CA, USA). The values for Bs and Bo were constrained using experimentally derived values. The IC₅₀ values were converted to equilibrium dissociation constants (K_i) [50,51]. Mean K_i values ± S.E.M. are reported for at least three independent experiments.