Fig. 6. BRET between GFP-G₂ and Kir3.1 is saturable and sensitive to receptor stimulation before surface targeting. (A) BRET saturation experiments in the ß₂AR stable line co-expressing G_s, Gß₁, GFP-G₂ and Kir3.1-RLuc or CD8-RLuc. The amount of plasmid encoding GFP-G₂ varied as represented by the ratio of fluorescence/luminescence whereas that for all the other plasmids was kept constant. Cells were treated (filled symbols) or not (open symbols) with 10 µM isoproterenol prior to assaying BRET. (B) Top: representative confocal image showing that Kir3.1-RLuc (green) has an intracellular distribution. Bottom: BRET experiments were performed using HEK 293 cells stably expressing the ß₂AR and transiently co-expressing GFP-Gß₁, G₂ and Kir3.1-RLuc. BRET was significantly increased (P<0.05) by the membrane-permeable agonist cimaterol but not by the membrane-impermeable agonist isoproterenol. Cimaterol-induced changes were blocked by pretreatment with propranolol. All ligands were used at a concentration of 1 µM. (C) Plasma membrane localization of Kir3.1 (green, middle panel) in the presence of co-expressed Flag-tagged Kir3.4 (red, top panel). Co-localization of Kir3.1 and Kir3.4 is demonstrated in the merged image (bottom panel). (D) BRET experiments were performed using the ß₂AR stable line co-expressing G_s, Gß₁, GFP-G₂ and Kir3.1-RLuc. Net agonist-stimulated BRET (mean ± s.d.) was significantly increased by isoproterenol (1 µM) if the cells co-expressed Kir3.4. Significant differences (*P<0.05) were determined by a paired t-test for three or more experiments in B and D.