Kinetics of M₁R/Gβ₁ interaction. (A) Cartoon of M₁R-CFP and Gβ₁-YFP constructs and cognate G proteins. (B) Confocal images of a pair of cells expressing M₁R-CFP and Gβ₁-YFP. Bar, 10 µm. (C) FRETr photometry time course for a single cell undergoing a 5-s exposure to 10 µM oxo-M. The top panel shows CFP_C fluorescence (blue trace, left axis) and YFP_C fluorescence (yellow trace, right axis), and the bottom panel shows the ratio, YFP_C/CFP_C (black). Sampling frequency: 2 Hz during baseline and 20 Hz during agonist. (D) Mean time course for 5-s exposures to oxo-M in six cells. Note the different time scales for onset and washout. Mean ± SEM (E) FRETr time course for a single cell. Oxo-M was stepped to different concentrations ranging from 10 nM to 50 µM as labeled. (F) FRETr concentration–response curve from steady-state values in E for six cells. Mean ± SEM.