Potentiation of Muscarinic M₃ Receptor Activation through a New Allosteric Site with a Novel Positive Allosteric...

Muscarinic M₃ (M₃) 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 M₃ receptors is not fully understood, presumably because of the lack of a potent and selective PAM. In this study, we investigated the pharmacological profile of ASP8302, a novel PAM of M₃ receptors, and explored the principal site of amino-acid sequences in the human M₃ receptor required for the potentiation of receptor activation. In cells expressing human M₃ and M₅ 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 M₃ 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, it identified threonine 230 as the amino acid essential for the PAM effect of ASP8302. These results demonstrate that ASP8302 enhances the activation of human M₃ receptors by interacting with a single amino acid distinct from the reported allosteric sites. Our findings suggest not only a novel allosteric site of M₃ receptors but also the potential application of ASP8302 to diseases caused by insufficient M₃ receptor activation. SIGNIFICANCE STATEMENT: The significance of this study is that the novel M₃ receptor positive allosteric modulator ASP8302 enhances the activation of human M₃ receptor by interacting with a residue distinct from the reported allosteric sites. The finding of Thr²³⁰ as a novel amino acid involved in the allosteric modulation of M₃ receptors provides significant insight into further research of the mechanism of allosteric modulation of M₃ and other muscarinic receptors.