Pharmacological characterization of the endocannabinoid sensor GRAB.

INTRODUCTION

The endocannabinoids (), 2-arachidonoylglycerol () and arachidonoyl ethanolamine (), are produced by separate enzymatic pathways, activate cannabinoid receptors with distinct pharmacology, and differentially regulate pathophysiological processes. The genetically encoded sensor, GRAB 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-cannabinoids 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-cannabinoid action.

METHODS

GRAB was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements.

RESULTS

2-AG increased GRAB fluorescent signal (EC = 85 nM), and the cannabinoid 1 receptor () antagonist, SR141617, decreased GRAB signal (, IC = 3.3 nM), responses that mirror their known potencies at cannabinoid 1 receptors (). GRAB fluorescent signal also increased in response to AEA (EC = 815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol ( and , ECs = 1.5 and 1.0 μM, respectively), Δ-tetrahydrocannabinol () and (ECs = 1.6 and 2.0 μM, respectively), and the artificial CBR agonist, CP55,940 (, EC = 82 nM); however their potencies were less than what has been described at CBR. Cannabidiol () did not affect basal GRAB fluorescent signal and yet reduced the 2-AG stimulated GRAB responses (IC = 8.8 nM).

CONCLUSIONS

2-AG and SR1 modulate the GRAB fluorescent signal with ECs that mirror their potencies at CBR whereas AEA, eCB analogues, THC and CP increase GRAB fluorescent signal with ECs significantly lower than their potencies at CBR. CBD reduces the 2-AG response without affecting basal signal, suggesting that GRAB retains the negative allosteric modulator () property of CBD at CBR. This study describes the pharmacological profile of GRAB to improve interpretation of changes in fluorescent signal in response to a series of known eCBs and CBR ligands.