(A) A computed molecular model of the M2 receptor (red) fused at the N-terminus to mCherry (yellow) and labeled with FlAsH (green) at a tetracysteine motif inserted in ECL2 between Val166 and Gly167. (B) Expanded views of ECL2 with the insert FCM and FlAsH. (C) M2 receptor bearing mCherry and the FlAsH-reactive insert (mCh-M2-FCM) was expressed in CHO cells and treated with FlAsH. Images were collected upon excitation at 498 nm and 0.37 µW. The emission spectrum from a single cell is shown in the figure (C). The spectrum was unmixed (Equation 10) to obtain the contributions of donor (kD, green) and acceptor (kA, red) to the fitted sum (black). (D) CHO cells expressing mCh-M2-FCM or the mCherry-tagged wild-type M2 receptor (mCh-M2) were treated with FlAsH and excited at 498 nm. The FRET efficiencies (Eapp) calculated for individual cells are shown as histograms, and the dashed lines depict the best fits of the Gaussian distribution (dark grey bars, mCh-M2-FCM, 31 cells, μ = 59.2 ± 1.2, σ = 7.7 ± 3.0; light grey bars, mCh-M2, 34 cells, μ = 0.50 ± 0.35, σ = 1.8 ± 0.5. (E) mCh-M2-FCM was expressed in CHO cells and reacted with FlAsH, and the value of Eapp was measured before and after addition of the inverse agonist NMS (1 µM, N = 26), the allosteric modulator gallamine (Gal, 10 mM, N = 42), NMS (1 µM) plus gallamine (10 mM, N = 18), the agonist carbachol (Car, 1 mM, N = 19), and the partial agonist pilocarpine (Pilo, 1 mM, N = 19). The ligand-dependent changes in Eapp are plotted in the figure (i.e., ∆Eapp ± S.D.), and the values are listed and compared in Figure 2—source data 1. The mean value of Eapp for cells in the absence of ligand was 60 ± 8% (N = 26).