Pharmacological Characterization of the Endocannabinoid Sensor GRABeCB2.0

Introduction: The endocannabinoids (eCBs), 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamine (AEA), are produced by separate enzymatic pathways, activate cannabinoid (CB) receptors with distinct pharmacological profiles, and differentially regulate pathophysiological processes. The genetically encoded sensor, GRABeCB2.0, detects real-time changes in eCB levels in cells in culture and preclinical model systems; however, its activation by eCB analogues produced by cells and by phyto-CBs remains uncharacterized, a current limitation when interpreting changes in its response. This information could provide additional utility for the tool in in vivo pharmacology studies of phyto-CB action. Materials and Methods: GRABeCB2.0 was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements. Results: 2-AG increased GRABeCB2.0 fluorescent signal (EC50=85 nM), and the cannabinoid 1 receptor (CB1R) antagonist, SR141716 (SR1), decreased GRABeCB2.0 signal (IC50=3.3 nM), responses that mirror their known potencies at the CB1R. GRABeCB2.0 fluorescent signal also increased in response to AEA (EC50=815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol (EC50=632 and 868 nM, respectively), Δ9-tetrahydrocannabinol (Δ9-THC), and Δ8-THC (EC50=1.6 and 2.0 μM, respectively), and the artificial CB1R agonist, CP55,940 (CP; EC50=82 nM); however their potencies were less than what has been described at CB1R. Cannabidiol (CBD) did not affect basal GRABeCB2.0 fluorescent signal and yet reduced the 2-AG stimulated GRABeCB2.0 responses (IC50=9.7 nM). Conclusions: 2-AG and SR1 modulate the GRABeCB2.0 fluorescent signal with EC50 values that mirror their potencies at CB1R, whereas AEA, eCB analogues, THC, and CP increase GRABeCB2.0 fluorescent signal with EC50 values significantly lower than their potencies at CB1R. CBD reduces the 2-AG response without affecting basal signal, suggesting that GRABeCB2.0 retains the negative allosteric modulator (NAM) property of CBD at CB1R. This study describes the pharmacological profile of GRABeCB2.0 to improve interpretation of changes in fluorescent signal in response to a series of known eCBs and CB1R ligands.