Radioligand binding assays

Binding assays were performed using Sf9 membrane fractions expressing the crystallization construct BRIL-KOR or HEK293 T membrane preparations transiently expressing KOR wt or KOR mutants. Binding assays were set up in 96-well plates in the standard binding buffer (50 mM Tris, 0.1 mM EDTA, 10 mM MgCl₂, 0.1% BSA, pH 7.40). Saturation binding assays with 0.1–20 nM [³H]-U69,593 in standard binding buffer were performed to determine equilibrium dissociation constant (Kd) and Bmax, whereas 10 µM final concentration of JDTic was used to define nonspecific binding. For the competition binding, 50 μL each of ³H-JDTic (final 0.8 nM), drug solution (3×) and homogenous membrane solution was incubated in 96-well plate in the standard binding buffer in the absence or presence of purified nanobodies (final concentration 5 µM). Reactions (either saturation or competition binding) were incubated for 2 h at room temperature in the dark and terminated by rapid vacuum filtration onto chilled 0.3% PEI-soaked GF/A filters followed by three quick washes with cold washing buffer (50 mM Tris HCl, pH 7.40) and read. Results (with or without normalization) were analyzed using GraphPad Prism 5.0 using one site—Fit Ki model (Eqs. (³) and (⁴)):

where X is the log molar concentration of unlabeled ligand; Y is the binding in any convenient units.

Top and Bottom are the plateaus in units of Y-axis; logK_i is the log of the equilibrium dissociation constant of tested ligand; [Hot] is the concentration of radioligand, and Kd is the equilibrium dissociation constant of radioligand.