(A) Concentration-response relation for the change in FRET in response to NE (filled squares) and Iso (open squares) in cells expressing the β₁-AR sensor (n = 4–6 per concentration and per group). (B) FRET signal caused by NE (10 μM; n = 10) is reversible by addition of the antagonist propranolol (Prop, 10 μM; n = 10) in HEK293 cells transiently expressing the β₁-AR sensor. (C) Comparison between the signaling properties of the β₁-AR sensor (n = 6) and the respective unlabeled receptor (n = 5) transiently expressed in HEK293 cells. cAMP detection was achieved using the Epac1-camps FRET sensor, and specific β₁-adrenergic stimulation was carried out by simultaneous application of ISO (1 nM) and the specific β₂-AR antagonist ICI 118551 (ICI; 200 nM). (D) Time-resolved changes in the FRET ratio F₅₃₅:F₄₈₀ of the β₁-AR sensor transiently expressed in HEK293 cells at various concentrations of NE. (E) Relationship between time activation constant (τ) and agonist concentration for the Gly389–β₁-AR sensor (filled circles; n = 6–14 per concentration) and for the Arg389–β₁-AR sensor (red circles; n = 5–8 per concentration). At low concentrations of agonist, τ values were directly proportional to the agonist concentration whereas at higher concentrations of agonist, the values approached a maximum of 60 ms. The Arg389 polymorphism did not affect the kinetics of receptor activation.