Fig. 3.

Analysis of receptor-G-protein interaction by FRET. FRET was measured between M₃-AChR-YFP and CFP-tagged Gγ₂. Excitation of CFP at 436 nm shifts the emission from 480 to 535 nm when the two fluorophores are close enough to permit FRET. A, HEK293 cells transiently expressing M₃-AChR-YFP together with CFP-Gγ₂ (and Gα_q and β₁) were superfused with 100 μM carbachol. This resulted in a rapid increase in FRET, as seen by the increase in the normalized FRET ratio (F_YFP/F_CFP). The increase in FRET was readily reversible upon agonist washout. B, upon superfusion with 100 μM carbachol, a decrease in CFP fluorescence and an increase in YFP fluorescence was observed. C, maximal receptor-G-protein interaction kinetics were compared with receptor-G-protein dissociation kinetics. Deactivation kinetics were analyzed similarly to activation kinetics. C, data for ACh (light gray) and carbachol (dark gray) for M₃-AChR-YFP (data represent means ± S.E. values; n = 9 for each column). D, concentration-response curves for the M₃-ACh receptor-G-protein interaction quantified by the amplitude of the ligand-evoked FRET changes. For ACh (□) EC₅₀ values were 0.14 ± 0.02 μM, whereas for carbachol (○) EC₅₀ values were 1.46 ± 0.26 μM, respectively (means ± S.E.; n = 10–12 for each ligand concentration). **, p < 0.01 (independent t test). AU, arbitary units.