Analysis of the Effects of Halothane on G<sub>i</sub>-Coupled Muscarinic M<sub>2</sub> Receptor Signaling in <i>Xenopus </i>Oocytes Using a Chimeric Gα Protein
Authors: Minami Kouichiro , Uezono Yasuhito , Shiraishi Munehiro , Okamoto Takashi , Ogata Jun-ichi , Horishita Takafumi , Taniyama Kohtaro , Shigematsu Akio 
Published: October 2004

Metabotropic G protein-coupled receptors have recently been recognized as targets for anesthetics and analgesics. In particular, G<sub>q</sub>-coupled receptors such as muscarinic M<sub>1</sub> receptors (M<sub>1</sub>R) 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 G<sub>i</sub>-coupled receptors. Here we report a method to analyze functions of G<sub>i</sub>-coupled receptors in <i>Xenopus</i> oocytes expressing a chimeric Gα protein. A chimeric Gα<sub>q</sub> protein Gα<sub>qi5</sub>, which contains carboxy-terminus five amino acids of Gα<sub>i</sub>, enables G<sub>i</sub>-coupled receptors to couple to Gq-coupled receptor-mediated downstream pathways such as activation of phospholipase C. We determined acetylcholine (ACh)-induced Ca<sup>2+</sup>-activated Cl<sup>–</sup> currents in <i>Xenopus </i>oocytes coexpressing G<sub>i</sub>-coupled muscarinic M<sub>2 </sub>receptors (M<sub>2</sub>R) with the chimeric Gα<sub>qi5</sub>. Although ACh did not induce any currents in oocytes expressing M<sub>2</sub>R alone, it caused robust Cl<sup>–</sup> currents in oocytes coexpressing M<sub>2</sub>R with Gα<sub>qi5</sub>. The EC<sub>50</sub> of the ACh-induced Cl<sup>–</sup> current mediated through Gα<sub>qi5</sub> was 0.2 µmol/l, which was 2.2 times higher than that of the ACh-induced G protein-activated inwardly rectifying K<sup>+</sup> currents activated by Gβγ subunits liberated from endogenously expressed Gα<sub>i</sub> in <i>Xenopus</i> oocytes. Other G<sub>i</sub>-coupled somatostatin type 2, 5-HT<sub>1A</sub> and δ-opioid receptors, when coexpressed with Gα<sub>qi5</sub> in oocytes, also caused robust Ca<sup>2+</sup>-activated Cl<sup>–</sup> currents. In oocytes coexpressing M<sub>2</sub>R and Gα<sub>qi5</sub>, a volatile anesthetic halothane inhibited M<sub>2</sub>R-induced Cl<sup>–</sup> currents in a concentration-dependent manner with the IC<sub>50</sub> of 1.1 mmol/l, suggesting that halothane inhibits M<sub>2</sub>R-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<sup>–</sup> currents induced by 1 µmol/l ACh in oocytes expressing M<sub>2</sub>R and G<sub>qi5</sub>. The rate of halothane-induced inhibition of Cl<sup>–</sup> currents elicited by ACh, however, was not changed in such oocytes pretreated with GF109203X. These findings suggest that halothane inhibits the M<sub>2</sub>R-induced signaling by acting at sites other than PKC activity. Collectively these findings suggest that the use of oocyte expressing Gα<sub>qi5</sub> would be helpful to examine the effects of anesthetics or analgesics on the function of G<sub>i</sub>-coupled receptors in the <i>Xenopus</i> oocyte expression system.