Fig. 3: M2 receptor activation results in an equilibrium of distinct M2R–G-protein signalling complexes in intact cells.
From: Ligand-specific activation trajectories dictate GPCR signalling in cells
Comparison of ligand-promoted changes in fluorescence intensity (ΔF/F0) of the M2R biosensors labelled with Tet–Cy3 at the indicated positions and stimulated with 1 mM ACh (grey), 100 µM iperoxo (green), 1 mM arecoline (purple) or 10 mM pilocarpine (pink) at endogenous G-protein levels or after GαoA(G203T) overexpression. a,b,d,e,g,h, Representative traces of the ΔF/F0 of single cells expressing the indicated M2R biosensors (M2R175 (a), M2R415 (b), M2R181 (d), M2R188 (e), M2R84 (g) and M2R414 (h)) at endogenous G-protein levels (left) or after GαoA(G203T) overexpression (right). The shaded areas indicate the duration of agonist superfusion, and the unshaded areas represent buffer application. c,f,i, Statistical summary of the normalized (norm.) changes in ΔF/F0 obtained from experiments in a and b (c), d and e (f), and g and h (i). Shown are the ligand-dependent differences in ΔF/F0 after GαoA(G203T) overexpression (Gmut) normalized to the mean ΔF/F0 of endogenous G-protein levels (Gendo, set to 0%). Negative values indicate decreases in ΔF/F0 after GαoA(G203T) overexpression, and positive values indicate increases in ΔF/F0 after GαoA(G203T) overexpression. Data are mean ± s.e.m., with each datapoint representing a single cell. M2R84 (iperoxo (15 cells examined over 5 independent experiments), ACh (14, 7), arecoline (10, 3), pilocarpine (20, 4)), M2R175 (iperoxo (11, 3), ACh (10, 3), arecoline (9, 3), pilocarpine (11, 3)), M2R181 (iperoxo (29, 4), ACh (41, 7), arecoline (34, 4), pilocarpine (20, 3)), M2R188 (iperoxo (16, 3), ACh (26, 4), arecoline (13, 3), pilocarpine (17, 3)), M2R414 (iperoxo (12, 4), ACh (31, 9), arecoline (10, 3), pilocarpine (16, 3)), M2R415 (iperoxo (28, 5), ACh (21, 6), arecoline (19, 5), pilocarpine (10, 3)). For c, f and i, P values were calculated using unpaired two-tailed t-tests. The constructs used were SP-M2RXXXTAG (Methods). The diagram in a was created in BioRender. Thomas, R. (2025) https://BioRender.com/loxqlqf.
