Competitive radioligand binding assays

In vitro binding assays were conducted on human opioid receptors stably transfected into CHO cells according to the published procedures²⁶. Binding assays were conducted on human opioid receptors stably transfected into CHO cells (CHO-hMOR, CHOhDOR, and CHO-hKOR) according to previously published procedures^26,40. Cell membranes were prepared as described previously²⁶, and stored at − 80 °C until use. Protein content of cell membrane preparations was determined by the method of Bradford using bovine serum albumin as the standard⁷⁷. Cell membranes (15–20 µg) were incubated in 50 mM Tris–HCl buffer (pH 7.4) with 1.1 nM [³H]DAMGO (MOR, K_d = 1.59 nM), 0.20 nM [³H]diprenorphine (DOR, K_d = 0.28 nM) or 1.2 nM [³H]U69,593 (KOR, K_d = 1.47 nM) in a final volume of 1 ml for 60 min at 25 °C. Non-specific binding was determined using 1–10 µM of the unlabeled counterpart of each radioligand. After incubation, reactions were terminated by rapid filtration through Whatman glass GF/C fiber filters. Filters were washed three times with 5 ml of ice-cold 50 mM Tris–HCl buffer (pH 7.4) using a Brandel M24R Cell Harvester (Gaithersburg, MD). Radioactivity retained on the filters was counted by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman Coulter Inc., Fullerton, CA). The inhibitory constant (K_i) values were calculated from the competition binding curves by nonlinear regression analysis and the Cheng-Prusoff eqaution⁷⁸. All experiments were performed in duplicate, and repeated at least three times with independently prepared samples.