Muscarinic M 3 (M 3 ) receptors mediate a wide range of acetylcholine (ACh)-induced functions, including visceral smooth muscle contraction and glandular secretion. Positive allosteric modulators (PAMs) can avoid various side effects of muscarinic agonists with their spatiotemporal receptor activation control and potentially better subtype selectivity. However, the mechanism of allosteric modulation of M 3 receptors is not fully understood, presumably due to the lack of a potent and selective PAM. In this study, we investigated the pharmacological profile of ASP8302, a novel PAM of M 3 receptors, and explored the principal site of amino acid sequences in the human M 3 receptor required for the potentiation of receptor activation. In cells expressing human M 3 and M 5 receptors, ASP8302 shifted the concentration-response curve (CRC) for carbachol to the lower concentrations with no significant effects on other subtypes. In a binding study with M 3 receptor-expressing membrane, ASP8302 also shifted the CRC for ACh without affecting the binding of orthosteric agonists. Similar shifts in the CRC of contractions by multiple stimulants were also confirmed in isolated human bladder strips. Mutagenesis analysis indicated no interaction between ASP8302 and previously reported allosteric sites; however, identified threonine 230 as the amino acid essential for the PAM effect of ASP8302. These results demonstrate that ASP8302 enhances the activation of human M 3 receptors by interacting with a single amino acid distinct from the reported allosteric sites. Our findings suggest not only a novel allosteric site of M 3 receptors but also the potential application of ASP8302 to diseases caused by insufficient M 3 receptor activation.

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