Muscarinic M₂ receptors directly activate G_q/11 and G_s G-proteins

Muscarinic M₂ receptors preferentially couple with the G _i/o class of G-proteins to inhibit cAMP synthesis. However, they can also stimulate net synthesis of cAMP and inositol phosphate (IP) accumulation. We investigated in intact Chinese hamster ovary (CHO) cells expressing human M₂ receptors (CHO-M₂ cells) whether direct interaction of M₂ receptors with G_s and G_q/11 G-proteins is responsible for the latter effects. Suppression of the G_sα subunit using RNA interference abolished stimulation of cAMP synthesis induced by 1 mM carbachol in both control and pertussis toxin-treated CHO-M₂ cells but had no effect on the inhibition of forskolin-stimulated cAMP synthesis. Carbachol stimulated accumulation of IP with an EC₅₀ of 79 μM. Removal of the G_q, G₁₁, or both α subunits reduced this response by 78, 54, and 92%, respectively, whereas suppression of the G_sα subunit had no effect. Similar results obtained in CHO cells expressing M₁ receptors that preferentially couple with G _s and G_q/11 G-proteins confirmed the efficiency of siRNA treatments. Stimulation of M₂ receptors in control and pertussis toxin-treated cells by a series of full agonists with respect to inhibition of adenylyl cyclase displayed different efficacies in stimulating IP accumulation. Carbachol, acetylcholine, and oxotremorine-M [N,N,N-trimethyl-4-(2-oxo-1- pyrolidinyl)-2-butyn-1-ammonium] behaved as full agonists, furmethide (N,N,N-trimethyl-2-furanmethammonium) and methylfurmethide [(5-methyl-2-furyl) methyltrimethylammonium] were partial agonists, and oxotremorine (1-[4-(1-pyrrolidinyl)-2-butynyl]-2-pyrrolidinone) had no effect. Our results provide direct evidence of M₂ receptor coupling with the α subunits of G_s and G_q/11 G-proteins and demonstrate induction of multiple receptor conformational states dependent on both the concentration and the nature of the agonist used.