Fig. 1: Structures and function of M2R ligands.
a Chemical structures of ACh, Ixo, and LY2119620. b ACh and Ixo competition curves of the M2R reconstituted into HDL particles in the presence or absence of GoA. Data are given as mean ± SEM from three independent experiments. c GTP-turnover of M2R bound to ACh or Ixo at different time points. Data are given as mean ± SEM from three or four independent experiments. d Ligand-dependent phosphorylation of M2R by GRK2 as a function of time. Intensity were extracted using Fiji75 from gel in Fig. S1B. e–h Concentration-response curves of ACh (e, g) and Ixo (f, h) toward G-protein activation and β-arrestin-2 recruitment in the presence of different concentrations of LY2119620. Dash lines indicate the maximal response of ACh and Ixo. Data with error bars are presented as mean ± SEM of three to fourteen independent experiments. i–l Statistics of Emax calculations for G-protein activation (i and k) and β-arrestin-2 recruitment (j, l). Emax values were analyzed by One-way ANOVA applying two-sided Dunnett’s multiple comparisons in PRISM 8.0. ***p < 0.001; **p < 0.01; *p < 0.05. m Effects of LY2119620 on G-protein activation efficacy of ACh and Ixo measured by GTPase GloTM assay. Statistical analyses were performed using the ordinary one-way ANOVA followed by the two-sided Sidak’s post-hoc test in PRISM 9.2.0. ***p < 0.001; *p < 0.05. Data with error bars are presented as mean ± SEM of three or five independent experiments.
