Comparison of cAMP and PKA signals at the OMM and cytosol. (A) Mixed populations of HEK cells expressing OMM-AKAR4 (two representative cells; red traces) or soluble AKAR4 (two representative cells; black traces). Both sensors responded to 10 nM isoproterenol (ISO); however, the OMM-AKAR4 signal reversed with significant delay compared with AKAR4 upon ISO removal (n = 4 experiments; 14 AKAR4 cells, 16 OMM-AKAR4 cells). (B) cAMP responses to ISO measured by OMM-EpacH90 (red trace; mean of four cells) or cytosolic EpacH90 (black trace) in HEK 293 cells (n = 3 experiments; 9 EpacH90 cells, 12 OMM-EpacH90 cells). (C) cAMP kinetics measured by OMM-EpacH90 (red trace; mean of three representative cells) or cytosolic EpacH90 (black trace; mean of four representative cells) in HeLa cells (n = 3 experiments; 10 EpacH90 cells, 19 OMM-EpacH90 cells). (D) Comparison of AKAR4 and OMM-AKAR4. Termination of PKA activity by agonist removal or H89 resulted in slower FRET reversal specifically at the OMM; two representative cells are depicted (n = 3 repeats; 12 AKAR4 cells, 14 OMM-AKAR4 cells).