The effects of isoflurane on native and chimeric muscarinic acetylcholine receptors: The role of protein kinase C

By using two electrode voltage clamps, we investigated the effects of isofl urane on m3 and chimeric m1/m3 muscarinic receptors and the role of protein kinase C (PKC) in the effects. Muscarinic receptors were expressed by inje ction of mRNA into Xenopus oocytes, and Ca2+-activated Cl- currents were me asured after the application of acetyl-beta -methylcholine. We constructed chimeric m1/m3 receptor DNA encoding the third intracellular loop of ml and the remainder from the m3 receptor. Chimeric and m3 receptors were inhibit ed by isoflurane, but the ml receptor was not. PKC activation with phorbol- 12-myrisate-13-acetate (50 nM) decreased signaling of both chimeric and m3 receptors significantly. Chelerythrine (20 muM, PKC inhibitor) abolished th e effect of isoflurane on chimeric and m3 signaling. Whereas isoflurane inh ibition of chimeric and m3 receptors was completely reversible after washou t with Tyrode's solution for 3 min, treatment with okadaic acid (500 nM, pr otein phosphatase inhibitor) rendered the inhibition irreversible. Taken to gether, 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.