Fig. 4: Ligand-specific activation kinetics of the MOR.
A, B Averaged single-cell FRET-based measurement of HEK293 cells stably expressing MOR conformation sensor. Cells were superfused with 100 µM of the indicated agonist, and measurements were recorded with a frequency of 10 Hz (A). After reaching a plateau, the applied agonist was washed off with extracellular buffer (indicated with the arrow) (B). C, D Half-time of activation (C) or deactivation (D) were evaluated by a one-phase mono-exponential fit based on (A, B) and Supplementary Fig. 4A–F (mean ± SEM, significance calculated against DAMGO, Brown–Forsyth & Welch’s ANOVA, P values: MetEnk C: 0.4637, D: 0.4374; Fentanyl C: <0.0001, D: 0.3526; Methadone C: 0.0056 D: 0.0006; Morphine C: <0.0001 D: 0.0174; Buprenorphine C: 0.0021 D: 0.1026). E Kinetics of deactivation were analyzed by a one-phase mono-exponential fit (mean ± SEM, significance calculated against DAMGO, Brown–Forsyth & Welch’s ANOVA, P values: MetEnk 0.0115; Fentanyl 0.1796; Methadone 0.0004; Morphine 0.0139; Buprenorphine 0.0006). F Kinetics of activation was calculated based on the kinetics of deactivation (E) and the EC50 of the respective agonist (Table 1); (mean ± SEM, significance calculated against DAMGO, Brown–Forsyth & Welch’s ANOVA, P values: MetEnk 0.4283; Fentanyl 0.0227; Methadone, Morphine, Buprenorphine: <0.0001). G The calculated Kon was plotted against the affinity of the respective agonist indicated by the pEC50 obtained in Fig. 2B.
