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Background and Purpose: Cannabichromene (CBC) is one of the most abundant phytocannabinoids in Cannabis spp. It has modest antinociceptive and antiinflammatory effects and potentiates some effects of Δ 9-tetrahydrocannabinol in vivo. How CBC exerts these effects is poorly defined and there is little information about its efficacy at cannabinoid receptors. We sought to determine the functional activity of CBC at cannabinoid CB 1 and CB 2 receptors. Experimental Approach: AtT20 cells stably expressing haemagglutinin-tagged human CB 1 and CB 2 receptors were used. Assays of cellular membrane potential and loss of cell surface receptors were performed. Key Results: CBC activated CB 2 but not CB 1 receptors to produce hyperpolarization of AtT20 cells. This activation was inhibited by a CB 2 receptor antagonist AM630, and sensitive to Pertussis toxin. Application of CBC reduced activation of CB 2 , but not CB 1 , receptors by subsequent co-application of CP55,940, an efficacious CB 1 and CB 2 receptor agonist. Continuous CBC application induced loss of cell surface CB 2 receptors and desensitization of the CB 2 receptor-induced hyperpolarization. Conclusions and Implications: CBC is a selective CB 2 receptor agonist displaying higher efficacy than tetrahydrocannabinol in hyperpolarizing AtT20 cells. CBC can also recruit CB 2 receptor regulatory mechanisms. CBC may contribute to the potential therapeutic effectiveness of some cannabis preparations, potentially through CB 2 receptor-mediated modulation of inflammation. 1 | INTRODUCTION Cannabichromene (CBC) is one of over 100 phytochemicals (collectively referred to as phytocannabinoids) that are found in Cannabis spp (ElSohly & Gul, 2014). CBC was identified in 1966 and is one of the most abundant phytocannabinoids alongside Δ 9-tetrahydrocannabinol (THC),

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The extracellular domains (ECDs) of Cys-loop receptors contain many aromatic amino acids, but only relatively few have been well studied. Here we explore the roles of Tyr and Trp residues in the ECD of the glycine receptor and show that four such residues that have not been previously studied (Y24, Y58, W170, and Y197) contribute significantly to the function of the protein. The residues were studied by creating mutant receptors, characterizing them using two-electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using structural information about the open and closed states of the receptor. Alanine substitution of all these residues ablates function or increases the glycine EC. There are also a number of changes in the relative maximal responses to taurine, a partial agonist, compared to glycine. Further mutations, in combination with structural information, suggest Y24 contributes to an anion-π interaction with a binding loop D residue, Y58 to an S-π interaction stabilizing the Cys loop, W170 to hydrophobic interactions stabilizing the hydrophobic interior of the subunit, and Y197 to a hydrogen bond linking binding loops B and C. These interactions appear to be broadly conserved in other Cys-loop receptors. Thus, we have identified new regions of the glycine receptor that are important contributors to receptor activation and are likely also to contribute to function in other members of this important protein family.

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