(A) Overall transmembrane topology of recombinant β₁-AR (Gly/Arg389–β₁-AR sensor) constructs. Using a linker (GlyGlyGlyGlyGly), we fused the cDNA encoding the Cerulean variant of GFP (Cer, blue) (49) to the carboxy terminus of the β₁-AR (position 477). YFP (yellow) was introduced into the third intracellular loop of the β₁-AR at position 273. (B) Visualization of transiently expressed β₁-AR sensor in HEK293 cells by confocal microscopy. Original magnification, ×63. (C) Effect of photobleaching. Emission intensities of YFP (535 nm, yellow) and Cerulean (Cer, 480 nm, blue) were recorded simultaneously from single cells expressing β₁-AR sensor using fluorescence microscopy. Emission intensities were recorded before and after the acceptor fluorophore was photobleached by 5 minutes exposure to light at 480 nm. (D) Time-resolved changes of the FRET ratio F₅₃₅/F₄₈₀ in single HEK293 cells expressing the β₁-AR sensor. Emission intensities of YFP (535 nm, yellow trace), Cer (480 nm, blue trace), and the FRET ratio F₅₃₅/F₄₈₀ (red trace) were recorded simultaneously from single cells. The decrease in FRET ratio is representative of the activation of the β₁-AR. The small arrow indicates the start of superfusion of the cell with 10 μM NE.