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.