Further characterization of the synergistic activation mechanism of cationic channels by M-2 and M-3 muscarinic receptors in mouse intestinal smooth muscle cells

In mouse ileal myocytes, muscarinic receptor-mediated cationic current (mI(cat)) occurs mainly through synergism of M-2 and M-3 subtypes involving G(i/o)-type GTP-binding proteins and phospholipase C (PLC). We have further studied the M-2/M-3 synergistic pathway. Carbachol-induced mI(cat) was markedly depressed by YM-254890, a G(q/11) protein inhibitor. However, the mI(cat) was unaffected by heparin, calphostin C, or chelerythrine, suggesting that mI(cat) activation does not involve signaling molecules downstream of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. M-2-knockout (KO) mice displayed a reduced mI(cat) (similar to 10% of wild-type mI(cat)) because of the lack of M2-Gi/o signaling. The impaired mI(cat) was insensitive to neuropeptide Y possessing a G(i/o)-stimulating activity. M-3-KO mice also displayed a reduced mI(cat) (similar to 6% of wild-type mI(cat)) because of the lack of M-3-G(q/11) signaling, and the mIcat was insensitive to prostaglandin F2 alpha possessing a G(q/11)-stimulating activity. These results suggest the importance of G(q/11)/PLC-hydrolyzed PIP2 breakdown itself in mI(cat) activation and also support the idea that the M-2/M-3 synergistic pathway represents a signaling complex consisting of M-2-G(i/o) and M-3-G(q/11)-PLC systems in which both G proteins are special for this pathway but not general in receptor coupling.