By using two electrode voltage clamps, we investigated the effects of isoflurane on m3 and chimeric m1/m3 muscarinic receptors and the role of protein kinase C (PKC) in the effects. Muscarinic receptors were expressed by injection of mRNA into Xenopus oocytes, and Ca(2+)-activated Cl(-) currents were measured after the application of acetyl-beta-methylcholine. We constructed chimeric m1/m3 receptor DNA encoding the third intracellular loop of m1 and the remainder from the m3 receptor. Chimeric and m3 receptors were inhibited by isoflurane, but the m1 receptor was not. PKC activation with phorbol-12-myrisate-13-acetate (50 nM) decreased signaling of both chimeric and m3 receptors significantly. Chelerythrine (20 microM, PKC inhibitor) abolished the effect of isoflurane on chimeric and m3 signaling. Whereas isoflurane inhibition of chimeric and m3 receptors was completely reversible after washout with Tyrode's solution for 3 min, treatment with okadaic acid (500 nM, protein phosphatase inhibitor) rendered the inhibition irreversible. Taken together, our results suggest that isoflurane inhibits m3 and chimeric m1/m3 muscarinic signaling by enhancing PKC activity and that the site of action is located outside of the third intracellular loop.
Implications: By use of the Xenopus oocyte expression system, we investigated the effects of isoflurane on muscarinic signaling and the role of protein kinase C in these effects. Our findings suggest that isoflurane inhibits muscarinic receptors through activation of protein kinase C and that the relevant phosphorylation sites are located outside the third intracellular loop.