A real time screening assay for cannabinoid CB1 receptor-mediated signaling

The cannabinoid CB1 receptor is expressed throughout the central nervous system where it functions to regulate neurotransmitter release and synaptic plasticity. While the CB1 receptor has been identified as a target for both natural and synthetic cannabinoids, the specific downstream signaling pathways activated by these various ligands have not been fully described. In this study, we developed a real-time membrane potential fluorescent assay for cannabinoids using pituitary AtT20 cells that endogenously express G protein-gated inward rectifier K⁺ (GIRK) channels and were stably transfected with the CB1 receptor using a recombinant lentivirus. In whole-cell patch clamp experiments application of the cannabinoid agonist WIN 55,212-2 to AtT20 cells expressing the CB1 receptor (AtT20/CB1) activated GIRK currents that were blocked by BaCl₂. WIN 55,212-2 activation of the GIRK channels was associated with a time- and concentration-dependent (EC₅₀ = 309 nM) hyperpolarization of the membrane potential in the AtT20/CB1 cells when monitored using a fluorescent membrane potential-sensitive dye. The WIN 55,212-2-induced fluorescent signal was inhibited by pretreatment of the cells with either the GIRK channel blocker tertiapin-Q or the CB1 receptor antagonist SR141716. The cannabinoids displayed a response of WIN 55,212-2 ≈ anandamide (AEA) > CP 55,940 > Δ⁹-tetrahydrocannabinol (THC) when maximal concentrations of the four ligands were tested in the assay. Thus, the AtT20/CB1 cell fluorescent assay will provide a straightforward and efficient methodology for examining cannabinoid-stimulated G_i signaling.