N6-substituted cAMP analogs inhibit bTREK-1 K+ channels and stimulate cortisol secretion by a protein kinase A-independent mechanism.
Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K+ channels whose inhibition by cAMP is coupled to membrane depolarization and cortisol secretion through complex signaling mechanisms. cAMP analogs with substitutions in the 6 position of the adenine ring selectively activate cAMP-dependent protein kinase (PKA) but not exchange proteins activated by cAMP (Epacs). In whole-cell patch-clamp recordings from AZF cells, we found that 6-benzoyl-cAMP (6-Bnz-cAMP) and 6-monobutyryl-cAMP potently inhibit bTREK-1 K+ channels, even under conditions in which PKA activity was abolished. Specifically, when applied through the patch electrode, 6-Bnz-cAMP inhibited bTREK-1 with an IC(50) of less than 0.2 microM. Inhibition of bTREK-1 by 6-Bnz-cAMP was not diminished by PKA antagonists, including N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), adenosine 3'-5'cyclic monophosphothiate, Rp-isomer, protein kinase inhibitor (PKI) (6-22) amide, and myristoylated PKI (14-22), applied alone or in combination, externally and intracellularly through the patch pipette. Under similar conditions, these same antagonists completely blocked PKA activation by 6-Bnz-cAMP. Inhibition of bTREK-1 by 6-Bnz-cAMP was voltage-independent and eliminated in the absence of ATP in the pipette solution. 6-Bnz-cAMP also produced delayed increases in cortisol synthesis and the expression of CYP11a1 mRNA that were only partially blocked by PKA antagonists. These results indicate that 6-Bnz-cAMP and other 6-substituted cAMP analogs can inhibit bTREK-1 K+ channels and stimulate delayed increases in cortisol synthesis by AZF cells through a PKA- and Epac-independent mechanism. They also suggest that adrenocorticotropin and cAMP function in these cells through a third cAMP-dependent protein. Finally, although 6-modified cAMP analogs exhibit high selectivity in activating PKA over Epac, they also may interact with other unidentified proteins expressed by eukaryotic cells.
Liu
Haiyan
H
Department of Neuroscience, OSU College of Medicine and Public Health, Columbus, OH, USA.
Enyeart
Judith A
JA
Enyeart
John J
JJ
D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000502:physiology*; D006854:Hydrocortisone / Q000378:metabolism*; D008297:Male; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects*; D024683:Potassium Channels, Tandem Pore Domain / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D014162:Transfection; D015383:Zona Fasciculata / Q000187:drug effects / Q000378:metabolism
D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024683:Potassium Channels, Tandem Pore Domain; C103742:potassium channel protein TREK-1; C050086:N(6)-benzoyl-cyclic AMP; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D006854:Hydrocortisone