Role of the cAMP sensor Epac as a determinant of K_ATP channel ATP sensitivity in human pancreatic β-cells and rat INS-1 cells

Abstract

Protein kinase A (PKA)-independent actions of adenosine 3′,5′-cyclic monophosphate (cAMP) are mediated by Epac, a cAMP sensor expressed in pancreatic β-cells. Evidence that Epac might mediate the cAMP-dependent inhibition of β-cell ATP-sensitive K⁺ channels (K_ATP) was provided by one prior study of human β-cells and a rat insulin-secreting cell line (INS-1 cells) in which it was demonstrated that an Epac-selective cAMP analogue (ESCA) inhibited a sulphonylurea-sensitive K⁺ current measured under conditions of whole-cell recording. Using excised patches of plasma membrane derived from human β-cells and rat INS-1 cells, we now report that 2′-O-Me-cAMP, an ESCA that activates Epac but not PKA, sensitizes single K_ATP channels to the inhibitory effect of ATP, thereby reducing channel activity. In the presence of 2′-O-Me-cAMP (50 μm), the dose–response relationship describing ATP-dependent inhibition of K_ATP channel activity (NP_o) is left-shifted such that the concentration of ATP producing 50% inhibition (IC₅₀) is reduced from 22 μm to 1 μm for human β-cells, and from 14 μm to 4 μm for rat INS-1 cells. Conversely, when patches are exposed to a fixed concentration of ATP (10 μm), the administration of 2′-O-Me-cAMP inhibits channel activity in a dose-dependent and reversible manner (IC₅₀ 12 μm for both cell types). A cyclic nucleotide phosphodiesterase-resistant ESCA (Sp-8-pCPT-2′-O-Me-cAMPS) also inhibits K_ATP channel activity, thereby demonstrating that the inhibitory actions of ESCAs reported here are unlikely to arise as a consequence of their hydrolysis to bioactive derivatives of adenosine. On the basis of such findings it is concluded that there exists in human β-cells and rat INS-1 cells a novel form of ion channel modulation in which the ATP sensitivity of K_ATP channels is regulated by Epac.