GTPγS Assay for CB1 Receptor Modulation

To assess the impact of 3c and 3d on G-protein coupling of CB₁, GTPγS assays were performed essentially as described previously.^{20 (link)} Membranes of CB₁ expressing cells were prepared, and 8 μg of membrane preparation was incubated with 0.1 μM of CP55,940 plus or minus the allosteric modulator, allosteric modulator alone, or 1 μM SR141716A alone, and 0.1 nM [³⁵S]GTPγS (1250 Ci/mmol; PerkinElmer Life Sciences, Boston, MA), 10 μM GDP (Sigma, St. Louis, MO), and 0.1% (w/v) BSA. GTPγS binding assay buffer (50 mM Tris-HCl, pH 7.4, 3 mM MgCl₂, 0.2 mM EGTA, and 100 mM NaCl) was added to 200 μL. The membranes were incubated for 1 h at 30°C. To determine nonspecific binding, 10 μM unlabeled GTPγS (Sigma) was used. To determine basal activity, membrane preparations were treated with vehicle (dimethylsulfoxide or DMSO) alone. Termination of the reaction was achieved through filtration using Whatman GF/C filter papers and washing with cold TME buffer. Bound radioactivity was measured by liquid scintillation counting.