PKA activity in the mitochondrial matrix measured using targeted FRET-based reporters. (A) Confocal images of HeLa cells expressing mito-AKAR4 loaded with MitoTracker red. (B) Mixed populations of HeLa cells transfected with mito-EpacH90 (black trace; mean of three cells) or mito-AKAR4 (red trace; mean of four cells) were initially bathed in Hepes-buffered solution and then switched to CO₂/HCO₃⁻-buffered solution (n = 5 experiments; 10 mito-AKAR4 cells, 12 mito-EpacH90 cells). (C) HeLa expressing mito-AKAR3 (two representative cells; red traces) or OMM-AKAR3 (two representative cells; black traces; n = 8; 20 OMM-AKAR3 cells, 20 mito-AKAR3 cells). (D) HeLa cells expressing OMM-AKAR3 (two representative cells; black traces) compared with neighboring mito-AKAR3 positive cells (two representative cells; red traces; n = 10 experiments; 24 OMM-AKAR3 cells, 28 mito-AKAR3 cells). (E) Cells expressing mito-AKAR3 (red trace; mean of three cells) or mito-EpacH90 plus mCherry (black traces; mean of six cells; typical of n = 4 experiments; 10 mito-AKAR3 cells, 16 mito-EpacH90 cells). (F) HEK cells transfected with mito-AKAR4 (red trace) or OMM-AKAR4 together with mCherry (black trace; mean of four cells); typical of n = 3 experiments; 6 OMM-AKAR4 cells, 8 mito-AKAR4 cells). Error bars indicate mean ± SD.