Fig. 5.

Prerequisites for reliable and reproducible measurements with FRET-based Epac sensors to detect G_i/o-protein-mediated cAMP decreases. For reliable and reproducible FRET measurements with FRET-based Epac sensors, achievement of steady-state conditions prior to application of different stimuli was of utmost importance. a, b Representative FRET measurement of HEK293 cells endogenously expressing β₂Rs (a) or over-expressing μRs (b) and the H74 construct. The time courses of the normalized yellow and cyan fluorescence signals (left) and of the normalized FRET signal (right) are displayed. Isoprenaline (200 μM, black bar, a) or forskolin (1 μM, FSK, black bar, b) are applied at steady-state conditions (time to steady state is indicated as white bar). The second stimulus DAMGO (100 nM, gray bar) is applied when steady-state conditions are achieved (time to steady state of FSK is indicated as a white bar, b). a, b Light gray bars indicate steady-state conditions used to determine the range of fluorescence changes as millivolts per second (left). c–e Summaries of the times to steady state of β₂R (c), α_2AR (d), or μR (e) and different indicated Epac sensor-expressing HEK293 cells. Results are displayed as boxplot analysis plus single values. Squares indicate mean values. Numbers over bars indicate the numbers of measured cells from at least 3 experimental days. f–h Slopes of the yellow and cyan florescence traces of β₂R (f), α_2AR (g), or μR (h) and different Epac sensor-expressing HEK293 cells determined before application of the first stimulus. *Fluorescence was detected as voltage of the transimpedance amplifier from the photodiode. i Summary of success rates of FRET measurements with HEK293 cells co-expressing different receptors and indicated FRET-based Epac sensors. Numbers over bars indicate the numbers of measured cells from at least 3 experimental days. j, k Analysis of the kinetics of FRET signal changes using the H74 (j) or the H187 sensor (k) after G_s- or G_i/o-coupled receptor activation calculated as exponential time constant as τ_1/2 displayed as boxplot analysis plus single values. Squares indicate mean values. Numbers indicate the numbers of measured cells from at least 3 experimental days