Exchange protein activated by cAMP (Epac) mediates cAMP-dependent but protein kinase A-insensitive modulation of vascular ATP-sensitive potassium channels

Exchange proteins directly activated by cyclic AMP (Epacs or cAMP-GEF) represent a family of novel cAMP-binding effector proteins. The identification of Epacs and the recent development of pharmacological tools that discriminate between cAMP-mediated pathways have revealed previously unrecognized roles for cAMP that are independent of its traditional target cAMP-dependent protein kinase (PKA). Here we show that Epac exists in a complex with vascular ATP-sensitive potassium (KATP) channel subunits and that cAMP-mediated activation of Epac modulates KATP channel activity via a Ca2+-dependent mechanism involving the activation of Ca2+-sensitive protein Phosphatase 2B (PP-2B, calcineurin). Application of the Epac-specific cAMP analogue 8-pCPT-2'-O-Me-cAMP, at concentrations that activate Epac but not PKA, caused a 41.6 +/- 4.7% inhibition (mean +/- S.E.M.; n = 7) of pinacidil-evoked whole-cell KATP currents recorded in isolated rat aortic smooth muscle cells. Importantly, similar results were obtained when cAMP was elevated by addition of the adenylyl cyclase activator forskolin in the presence of the structurally distinct PKA inhibitors, Rp-cAMPS or KT5720. Activation of Epac by 8-pCPT-2'-O-Me-cAMP caused a transient 171.0 +/- 18.0 nM (n = 5) increase in intracellular Ca2+ in Fura-2-loaded aortic myocytes, which persisted in the absence of extracellular Ca2+. Inclusion of the Ca2+-specific chelator BAPTA in the pipette-filling solution or preincubation with the calcineurin inhibitors, cyclosporin A or ascomycin, significantly reduced the ability of 8-pCPT-2'-O-Me-cAMP to inhibit whole-cell KATP currents. These results highlight a previously undescribed cAMP-dependent regulatory mechanism that may be essential for understanding the physiological and pathophysiological roles ascribed to arterial KATP channels in the control of vascular tone and blood flow.