Analysis of the Effects of Halothane on Gi-Coupled Muscarinic M2 Receptor Signaling in Xenopus Oocytes Using a Chimeric Gα Protein

Metabotropic G protein-coupled receptors have recently been recognized as targets for anesthetics and analgesics. In particular, Gq-coupled receptors such as muscarinic M1 receptors (M1R) and 5-hydroxytryptamine (5-HT) type 2A receptors have been reported to be targets for anesthetics. Much less is known, however, about the effects of anesthetics on Gi-coupled receptors. Here we report a method to analyze functions of Gi-coupled receptors in Xenopus oocytes expressing a chimeric Gα protein. A chimeric Gαq protein Gαqi5, which contains carboxy-terminus five amino acids of Gαi, enables Gi-coupled receptors to couple to Gq-coupled receptor-mediated downstream pathways such as activation of phospholipase C. We determined acetylcholine (ACh)-induced Ca2+-activated Cl– currents in Xenopus oocytes coexpressing Gi-coupled muscarinic M2 receptors (M2R) with the chimeric Gαqi5. Although ACh did not induce any currents in oocytes expressing M2R alone, it caused robust Cl– currents in oocytes coexpressing M2R with Gαqi5. The EC50 of the ACh-induced Cl– current mediated through Gαqi5 was 0.2 µmol/l, which was 2.2 times higher than that of the ACh-induced G protein-activated inwardly rectifying K+ currents activated by Gβγ subunits liberated from endogenously expressed Gαi in Xenopus oocytes. Other Gi-coupled somatostatin type 2, 5-HT1A and δ-opioid receptors, when coexpressed with Gαqi5 in oocytes, also caused robust Ca2+-activated Cl– currents. In oocytes coexpressing M2R and Gαqi5, a volatile anesthetic halothane inhibited M2R-induced Cl– currents in a concentration-dependent manner with the IC50 of 1.1 mmol/l, suggesting that halothane inhibits M2R-induced cellular responses at clinically relevant concentrations. Treatment with the protein kinase C inhibitor GF109203X produced a 3.5-fold enhancement of the initial Cl– currents induced by 1 µmol/l ACh in oocytes expressing M2R and Gqi5. The rate of halothane-induced inhibition of Cl– currents elicited by ACh, however, was not changed in such oocytes pretreated with GF109203X. These findings suggest that halothane inhibits the M2R-induced signaling by acting at sites other than PKC activity. Collectively these findings suggest that the use of oocyte expressing Gαqi5 would be helpful to examine the effects of anesthetics or analgesics on the function of Gi-coupled receptors in the Xenopus oocyte expression system.