Pharmacological Characterization of the Endocannabinoid Sensor GRAB(eCB2.0).

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, GRAB(eCB2.0), 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: GRAB(eCB2.0) was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements. Results: 2-AG increased GRAB(eCB2.0) fluorescent signal (EC(50)=85 nM), and the cannabinoid 1 receptor (CB(1)R) antagonist, SR141716 (SR1), decreased GRAB(eCB2.0) signal (IC(50)=3.3 nM), responses that mirror their known potencies at the CB(1)R. GRAB(eCB2.0) fluorescent signal also increased in response to AEA (EC(50)=815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol (EC(50)=632 and 868 nM, respectively), Δ(9)-tetrahydrocannabinol (Δ(9)-THC), and Δ(8)-THC (EC(50)=1.6 and 2.0 μM, respectively), and the artificial CB(1)R agonist, CP55,940 (CP; EC(50)=82 nM); however their potencies were less than what has been described at CB(1)R. Cannabidiol (CBD) did not affect basal GRAB(eCB2.0) fluorescent signal and yet reduced the 2-AG stimulated GRAB(eCB2.0) responses (IC(50)=9.7 nM). Conclusions: 2-AG and SR1 modulate the GRAB(eCB2.0) fluorescent signal with EC(50) values that mirror their potencies at CB(1)R, whereas AEA, eCB analogues, THC, and CP increase GRAB(eCB2.0) fluorescent signal with EC(50) values significantly lower than their potencies at CB(1)R. CBD reduces the 2-AG response without affecting basal signal, suggesting that GRAB(eCB2.0) retains the negative allosteric modulator (NAM) property of CBD at CB(1)R. This study describes the pharmacological profile of GRAB(eCB2.0) to improve interpretation of changes in fluorescent signal in response to a series of known eCBs and CB(1)R ligands.