@article {Liu1290, author = {Haiyan Liu and Judith A. Enyeart and John J. Enyeart}, title = {N6-Substituted cAMP Analogs Inhibit bTREK-1 K+ Channels and Stimulate Cortisol Secretion by a Protein Kinase A-Independent Mechanism}, volume = {76}, number = {6}, pages = {1290--1301}, year = {2009}, doi = {10.1124/mol.109.057075}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {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 IC50 of less than 0.2 μM. 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{\textendash}22) amide, and myristoylated PKI (14{\textendash}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.{\textcopyright} 2009 The American Society for Pharmacology and Experimental Therapeutics}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/76/6/1290}, eprint = {https://molpharm.aspetjournals.org/content/76/6/1290.full.pdf}, journal = {Molecular Pharmacology} }