Fig. 3.

FRET-based cAMP sensors reliably detect cAMP level decreases induced by activation of G_i/o-protein-coupled μ opioid receptors. FRET measurements with HEK293 cells expressing G_i/o-protein-coupled μ opioid receptors together with one of the indicated FRET-based Epac constructs. a–f Representative FRET measurements are displayed showing time courses of the normalized yellow and cyan fluorescence signals (left) and of the normalized FRET signal (right). Black bars indicate application of the adenylyl cyclase activator forskolin (1 μM, FSK) in submaximal concentration to increase basal cAMP levels (a–e). Gray bars show application of the selective μ receptor agonist DAMGO (100 nM). g Summary of FRET signal increases induced by DAMGO after forskolin pre-stimulation in the presence (hatched bars) or absence (solid bars) of the selective μ receptor antagonist CTAP (500 nM). Numbers over bars indicate the numbers of measured cells and the number of individual coverslips from at least 3 experimental days. h Summary of FRET signal increases induced by 100 nM DAMGO in the presence (hatched bars) or absence (solid bars) of CTAP (500 nM). Right insets show representative FRET signal trace with application of increasing concentrations of DAMGO (top) and the concentration response curve displayed as mean ± s.e.m. of three independent measurements (bottom). The curve was fitted using the Hill equation. Numbers over bars indicate the numbers of measured cells and the number of individual cover slips from at least 3 experimental days. Significances tested between cells treated and not treated with CTAP. *P < 0.05, **P < 0.01, ***P < 0.001