Role of the cAMP sensor Epac as a determinant of KATP channel ATP sensitivity in human pancreatic beta-cells and rat INS-1 cells.
Protein kinase A (PKA)-independent actions of adenosine 3',5'-cyclic monophosphate (cAMP) are mediated by Epac, a cAMP sensor expressed in pancreatic beta-cells. Evidence that Epac might mediate the cAMP-dependent inhibition of beta-cell ATP-sensitive K(+) channels (K(ATP)) was provided by one prior study of human beta-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 beta-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 microM), 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 (IC(50)) is reduced from 22 microM to 1 microM for human beta-cells, and from 14 microM to 4 microM for rat INS-1 cells. Conversely, when patches are exposed to a fixed concentration of ATP (10 microM), the administration of 2'-O-Me-cAMP inhibits channel activity in a dose-dependent and reversible manner (IC(50) 12 microM 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 beta-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.
Kang
Guoxin
G
Department of Physiology, New York University School of Medicine, New York, NY, USA
Leech
Colin A
CA
Chepurny
Oleg G
OG
Coetzee
William A
WA
Holz
George G
GG
D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D054086:KATP Channels / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D015398:Signal Transduction
D020662:Guanine Nucleotide Exchange Factors; D054086:KATP Channels; C116122:RAPGEF3 protein, human; C526973:Rapgef3 protein, rat; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases