Fig. 5: Characterization of the M4AChR selective modulators.
a Amino-acid sequence alignment of MT3 with identified M4AChR-selective variants. Residue numbers are labeled above the alignment. Residues that differ from MT3 are highlighted in red. b Binding activity of M4S3 to α1-, α2- and mAChR-family receptors. HEK293 cells expressing the indicated receptors were stained with 1 μM M4S3 and analyzed by flow cytometry. Data represent means ± SEM (n = 3). c Comparison of the affinity between M4S3 and MT3 for M4AChR. HEK293 cells expressing M4AChR were stained with M4S3 or MT3 at the indicated concentrations. The data were normalized as the percentage of the maximal M4AChR binding and presented as the mean ± SEM (n = 3). d Antagonist activity of M4S3 measured by NanoBiT assays. The cells expressing the NanoBiT–G protein and M4AChR were treated with titrated M4S3 or MT3. Luminescent signals were measured before and after ligand stimulation (10 μM bethanechol chloride). Data were normalized to the maximum luminescent signals of the M4AChR and presented as the mean ± SEM (n = 3). e Measurement of the binding affinity of finger loop 1-only variants to M4AChR. Amino-acid sequence alignment of MT3 with identified finger loop 1-only variants (left). Residue numbers are labeled. Residues that differ from MT3 are highlighted in red. HEK293 cells expressing the M4AChR were stained with the variants at the indicated concentrations. The data were normalized as the percentage of the maximal M4AChR binding and presented as the mean ± SEM (n = 3). f Binding of M4S3 and MT3 to chimeric M4AChR and M2AChR receptors. HEK293 cells expressing the chimeric receptors were stained with M4S3 or MT3 at a concentration of 1 μM. The data were normalized as the percentage of the maximal M4AChR binding and presented as the mean ± SEM (n = 3). Binding affinity values are provided in Supplementary Table 2d, 2f, antagonist activity values are provided in Supplementary Table 3. Source data are provided in the Source Data file.
