Kinetics of Gα_q/Gβ₁ separation. All cells coexpress M₁R, Gα_q-CFP, Gβ₁-YFP, Gγ₂, and GRK2, except GRK2 is absent in one part of D. (A) Cartoon of Gα_q-CFP and Gβ₁-YFP constructs and cognate G proteins. (B) Confocal images of a group of cells expressing Gα_q-CFP and Gβ₁-YFP in the presence of GRK2. Bar, 10 µm. (C) FRETr photometry time course for a single cell undergoing a 10-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 10 Hz during agonist. (D) Mean time course for 10-s exposures to oxo-M in 10 cells in the absence (open circles) and 8 cells in the presence (closed circles) of GRK2. Note the different time scales for onset and washout. Mean ± SEM. For clarity in display, points were pooled in 500-ms bins for onset and 4-s bins for washout. (E) FRETr time course for a single cell. Oxo-M was stepped to different concentrations ranging from 1 nM to 10 µM as labeled. For clarity in display, trace is smoothed. (F) FRETr concentration–response curve from steady-state values in E for six cells. Mean ± SEM.