EXPLORING ALLOSTERIC MODULATION OF MUSCARINIC M5 RECEPTOR VIA SITE-DIRECTED MUTAGENESIS

The muscarinic acetylcholine receptor (mAChR) family comprises five subtypes (M1–M5) of class A G-protein coupled receptors (GPCRs), broadly expressed in the nervous system. Among these, M5 has emerged as a promising therapeutic target due to its selective localization in dopaminergic neurons of the ventral tegmental area (VTA), a region implicated in reward processing and neuropsychiatric disorders such as schizophrenia and substance use disorder. The high conservation of the orthosteric binding site across mAChRs complicates subtype-selective drug design, prompting interest in allosteric modulation. Structural studies have proposed three distinct allosteric sites on M5, including the extracellular vestibule and two additional pockets accommodating modulators such as the positive allosteric modulator (PAM) VU6007678 and the negative allosteric modulator (NAM) ML375. Here, we systematically introduced single-point mutations within these sites and assessed their functional impact using calcium mobilization assays exploiting Gq-mediated signalling. A matrix-style design enabled simultaneous titration of agonist and modulator, revealing key residues essential for allosteric ligand binding and receptor activity. Our findings provide functional validation of structural models and demonstrate that these sites are not fully conserved across the mAChR family. This approach establishes a framework for high-throughput characterization of M5-selective modulators, advancing efforts toward targeted therapies for neuropsychiatric disorders.