Signalling at CB₂ receptor and naturally occurring CB₂ SNPs variants

CB₂, one of the key components of the endocannabinoid system is emerging as a potential drug target for the treatment of various pathological conditions, especially pain and inflammation. However, there is little available quantitative data about the relative efficacy of CB₂ selective ligands. Moreover, several single nucleotide polymorphisms (SNPs) of the CB₂ appear in the human populations may influence drug efficacy or alter the signalling of CB₂. Thus, this study aimed to find out a suitable CB₂ irreversible antagonist that could be used to determine ligand efficacy precisely and investigate the ligand responses in six CB₂ variants. 

CB₁, CB₂ and six CB₂ variants (63Q, 63R, 63Q/L133I, 63R/ L133I, 63Q/H316Y, 63R/H316Y) were stably transfected in mouse pituitary AtT20 FlpIn cells. CB₁ and CB₂ cell lines were used to determine the pharmacological property of three compounds AM1336, AM1335, and AM10257 while the CB₂ variants were used to investigate the ligand responses. FLIPR membrane potential dye was used to measure the ligand induced changes in hyperpolarization in cells in the response to GIRK channel activation. Receptor depletion assay was performed in cells expressing 63Q and 63R variants and the ligand efficacy was calculated by fitting the data in the operational model of agonism using non‐linear regression. 

All the three-compounds tested were CB₁ agonists but CB₂ irreversible antagonists. AM1336 appeared to be a low efficacy CB₁ agonist and its effect was completely reversed by a CB₁ specific antagonist SR141716. While AM1336 was cannabinoid receptor-specific, 10 μM AM1335 and AM10257 decreased the maximum response of somatostatin by 33 ± 1% and 38 ± 2% respectively in wild type AtT20 cells. AM1336 was proved as an effective tool for efficacy analysis of CB₂ selective ligands as it depleted more than 80% of receptors in cell lines expressing 63Q and 63R variants. No significant differences in ligand responses were observed among the CB₂ SNPs variants. 

In conclusion, AM1336 could be useful to determine ligand efficacy quantitatively at CB₂ -SNPs in future.