Fig. 5.

Analysis of G-protein activation by FRET. FRET was measured between YFP-tagged Gα_q 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 YFP-tagged Gα_q together with CFP-Gγ₂ (and M₃-AChR and β₁) were superfused with 100 μM carbachol or 100 μM ACh. This resulted in a rapid decrease in FRET as seen by the decrease in the normalized FRET ratio (F_YFP/F_CFP). The decrease in FRET was readily reversible upon agonist washout. B, upon superfusion with 100 μM carbachol or 100 μM ACh, an increase in CFP fluorescence and a decrease in YFP fluorescence were observed. C, maximal G-protein activation kinetics for ACh (light gray bars) and carbachol (dark gray bars) were compared with G-protein deactivation kinetics. Deactivation kinetics were analyzed similarly to activation kinetics (data represent means ± S.E. values; n = 18 for ACh and n = 17 for carbachol). AU, arbitary units.