Angiotensin II inhibits native bTREK-1 K+ channels through a PLC-, kinase C-, and PIP2-independent pathway requiring ATP hydrolysis

Abstract

Angiotensin II (ANG II) inhibits bTREK-1 (bovine KCNK2) K+ channels in bovine adrenocortical cells through a Gq-coupled AT1 receptor by activation of separate Ca2+- and ATP hydrolysis-dependent signaling pathways. Whole cell patch-clamp recording from bovine adrenal zona fasciculata (AZF) cells was used to characterize the ATP-dependent signaling mechanism for inhibition of bTREK-1 by ANG II. We discovered that ATP-dependent inhibition of bTREK-1 by ANG II occurred through a novel mechanism that was independent of PLC and its established downstream effectors. The ATP-dependent inhibition of bTREK-1 by ANG II was not reduced by the PLC antagonists edelfosine and U73122, or by the PKC antagonists bisindolylmaleimide I (BIM) or calphostin C. bTREK-1 was partially inhibited (∼25%) by the PKC activator phorbol 12,13 dibutyrate (PDBu) through an ATP-dependent mechanism that was blocked by BIM. Addition of Phosphatidylinositol( 4 , 5 ) bisphosphate diC8 [DiC8PI( 4 , 5 )P2], a water-soluble derivative of phosphotidyl inositol 4,5 bisphosphate (PIP2) to the pipette solution failed to alter inhibition by ANG II. bTREK-1 inhibition by ANG II was also insensitive to antagonists of other protein kinases activated by ANG II in adrenocortical cells but was completely blocked by inorganic polytriphosphate PPPi. DiC8PI( 4 , 5 )P2 was a weak activator of bTREK-1 channels, compared with the high-affinity ATP analog N6-(2-phenylethyl)adenosine-5′-O-triphosphate (6-PhEt-ATP). These results demonstrate that the modulation of bTREK-1 channels in bovine AZF cells is distinctive with respect to activation by phosphoinositides and nucleotides and inhibition by Gq-coupled receptors. Importantly, ANG II inhibits bTREK-1 channels through a novel pathway that is different from that described for inhibition of native TREK-1 channels in neurons, or cloned channels expressed in cell lines. They also indicate that, under physiological conditions, ANG II inhibits bTREK-1 and depolarizes AZF cells by two, novel, independent pathways that diverge proximal to the activation of PLC.