Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K+ channels that belong to the two-pore/four-transmembrane segment (2P/4TMS) family of K+ channels. They set the resting membrane potential and function in coupling receptor activation at the membrane to depolarization-dependent Ca2+ entry and cortisol secretion. Angiotensin II (ANG II) and adrenocorticotropic hormone (ACTH) inhibit bTREK-1 channels in AZF cells. However, the signaling pathways underlying the inhibition of bTREK-1 by ANG II and ACTH are partially understood.
In my thesis, I explored the signaling mechanisms by which ANG II and ACTH inhibit bTREK-1 channels and bTREK-1 pharmacology from bovine AZF cells in whole cell patch clamp recordings. I found that the ATP-dependent inhibition of bTREK-1 by ANG II occurred through a novel mechanism that was independent of phospholipase C (PLC), protein kinase C (PKC) and phosphatidylinositol 4, 5 bisphosphate (PIP2). These results indicate that under physiological conditions, ANG II inhibits bTREK-1 by two novel independent pathways that diverge proximal to the activation of PLC, which is different from that described in expression systems.
Next, ACTH, NPS-ACTH, and forskolin inhibited bTREK-1 channels in AZF cells through cAMP by both PKA-dependent and independent signaling mechanisms which involved the activation of Epac2 (a guanine nucleotide exchange protein activated by cAMP). These results indicate that ACTH and cAMP inhibit bTREK-1 through parallel PKA-and Epac-dependent signaling mechanisms which may provide for failsafe membrane depolarization by ACTH.
Finally, I investigated five Ca2+ channel antagonists as inhibitors of bTREK-1 channels and discovered that selected dihydropyridine (DHP) Ca2+ channel antagonists such as amlodipine and niguldipine potenly inhibited native bTREK-1 channels. Overall, these results demonstrate that organic Ca2+ antagonists rank as the most potent inhibitors of TREK-1 channels yet described.
Together, these studies described novel signaling mechanisms by which ANG II and ACTH inhibit bTREK-1 channels in AZF cells and established the pharmacological profile of TREK-1 channels. These results provide important insights on the physiological roles of bTREK-1 channels in cortisol secretion and the potential target for drug development.