Cannabinoid receptor characterization in Xenopus oocytes

These studies focused on evaluating receptor pharmacology, structure-activity, and desensitization in a system demonstrating cannabinoid receptor coupling to G-protein gated inwardly rectifying potassium (GIRK) channels. Cannabinoids were tested on Xenopus oocytes co-expressing the CB 1 receptor and GIRK1 and GIRK4 channels (CB1/GIRK1/4) or the CB2 receptor and GIRK1/4 channels (CB2/GIRK1/4). Cannabinoids enhanced currents carried by GIRK channels in the CB1/GIRK1/4 and CB2/GIRK1/4 system, however, the CB2 receptor did not couple efficiently to GIRK1/4 channels. The CB1 selective antagonist, SR141716A, alone acted as an inverse agonist by inhibiting GIRK currents in oocytes expressing CB1/GIRK1/4. We also observed marked desensitization of CB1 receptor function when multiple concentration of CP 55,940 were cumulatively applied to oocytes expressing CB1/GIRK1/4. This same effect was not seen with WIN 55,212-2 under the same condition.; A conserved aspartate residue, which was previously shown to be critical for G- protein coupling in cannabinoid receptors, was mutated to asparagine (D163N) and analyzed. Oocytes co-expressing CB1/GIRK1/4 or D163N/GIRK1/4 were compared. The potency of WIN 55,212-2 at the mutant receptor was similar to wild type, however, its efficacy was substantially reduced. CP 55,940 did not elicit currents in oocytes expressing D163N/GIRK1/4.; A recently constructed molecular model implicates a conserved tyrosine residue (Y275) in the fifth transmembrane domain as part of the binding site for cannabinoids. Y275 was mutated to phenylalanine or isoleucine in order to determine the role of this amino acid. A stable cell line transfected with the mutant receptor cDNAs was evaluated in radioligand binding and the mutant receptors were co-expressed with GIRK1/4 in Xenopus oocytes to assess functional coupling. Substitution of phenylalanine for Y275 resulted in a receptor with essentially wild-type properties. Substitution of isoleucine for Y275 produced receptor with an altered ligand recognition profile and a loss of signal transduction.; In summary, it appears the CB1 and CB2 receptors couple differently to GIRK1/4 channels. In the CB1/GIRK1/4 system, cannabinoid receptors demonstrated the ability to enhance or inhibit GIRK currents and were susceptible to the process of desensitization. Furthermore, amino acid residues were identified in the CB1 receptor which are required for normal communication with GIRK channels.