Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin

Abstract

Bovine adrenocortical cells express bTREK-1 K ϩ (bovine KCNK2) channels that are inhibited by ANG II through a Gq-coupled receptor by separate Ca 2ϩ and ATP hydrolysis-dependent signaling pathways. Whole cell and single patch clamp recording from adrenal zona fasciculata (AZF) cells were used to characterize Ca 2ϩ -dependent inhibition of bTREK-1. In whole cell recordings with pipette solutions containing 0.5 mM EGTA and no ATP, the Ca 2ϩ ionophore ionomycin (1 M) produced a transient inhibition of bTREK-1 that reversed spontaneously within minutes. At higher concentrations, ionomycin (5-10 M) produced a sustained inhibition of bTREK-1 that was reversible upon washing, even in the absence of hydrolyzable [ATP] i. BAPTA was much more effective than EGTA at suppressing bTREK-1 inhibition by ANG II. When intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) was buffered to 20 nM with either 11 mM BAPTA or EGTA, ANG II (10 nM) inhibited bTREK-1 by 12.0 Ϯ 4.5% (n ϭ 11) and 59.3 Ϯ 8.4% (n ϭ 4), respectively. Inclusion of the water-soluble phosphatidylinositol 4,5-bisphosphate (PIP 2) analog DiC8PI(4,5)P2 in the pipette failed to increase bTREK-1 expression or reduce its inhibition by ANG II. The open probability (P o) of unitary bTREK-1 channels recorded from inside-out patches was reduced by Ca 2ϩ (10 -35 M) in a concentration-dependent manner. These results are consistent with a model in which ANG II inhibits bTREK-1 K ϩ channels by a Ca 2ϩ -dependent mechanism that does not require the depletion of membrane-associated PIP 2. They further indicate that the Ca 2ϩ source is located in close proximity within a "Ca 2ϩ nanodomain" of bTREK-1 channels, where [Ca 2ϩ ]i may reach concentrations of Ͼ10 M. bTREK-1 is the first two-pore K ϩ channel shown to be inhibited by Ca 2ϩ through activation of a G protein-coupled receptor. adrenocortical; potassium channel; ionomycin; phosphatidylinositol 4,5-bisphosphate; nanodomain BOVINE ADRENOCORTICAL CELLS including cortisol-secreting zona fasciculata (AZF) cells and aldosterone-secreting zona glomerulosa (AZG) cells express bTREK-1 (bovine KCNK2) leak-type K ϩ channels that function pivotally in the physiology of corticosteroid hormone secretion (14,17,18,35). These two-pore, four-transmembrane spanning (2P/4TMS) K ϩ channels set the resting membrane potential of bovine adrenocortical cells and are inhibited by ANG II at concentrations identical to those that trigger membrane depolarization and corticosteroid secretion (14,35).The signaling pathways that couple ANG II receptors to bTREK-1 inhibition are complex and only partially understood. In both AZF and AZG cells, ANG II inhibits bTREK-1 through activation of a Gq-coupled AT 1 receptor (14,35,36). AT 1 receptors in these adrenocortical cells have been linked to the activation of multiple signaling pathways (28,29,45,50). However, the Gq-dependent activation of PLC␤, leading to the hydrolysis of membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and the production of inositol 1,4,5-trisphosphate (IP 3 ) and diacylglyc...

Differential phospholipase C‐dependent modulation of TASK and TREK two‐pore domain K+ channels in rat thalamocortical relay neurons

“…; Enyeart et al . ). The findings presented here are in good agreement with the activation of TREK current by PIP 2 and other phospholipids like PIP (Chemin et al .…”

“…For TREK-1 it has been shown that a decrease in membrane PIP 2 levels promotes channel closing via protein kinase A and that the membrane potential regulation of the channel is sensitive to PIP 2 (Chemin et al 2005(Chemin et al , 2007Lopes et al 2005Lopes et al , 2007Segal-Hayoun et al 2010). Alternatively TREK-1 inhibition by other components of the G αq /PLCβ pathway, namely PKC, Ca 2+ and DAG, has been suggested (Chemin et al 2003;Murbartian et al 2005;Enyeart et al 2011). The findings presented here are in good agreement with the activation of TREK current by PIP 2 and other phospholipids like PIP (Chemin et al 2005;Segal-Hayoun et al 2010).…”

TREK-1 currents in smooth muscle cells from pregnant human myometrium

“…The activity of the TREK-1 channel has been shown to be regulated by numerous factors, including AA, pH, stretch, phosphorylation, nitric oxide, temperature, and others (20,(22)(23)(24)57). Here we provide provocative evidence that pregnancy regulates the channel, such that cells from nonpregnant women do not activate when stimulated by AA, despite expression of the channel (13), albeit at a lower level than during pregnancy.…”

Ca2+ and K+ channels of normal human adrenal zona fasciculata cells: Properties and modulation by ACTH and AngII

“…In this regard, in bovine AZF cells, AngII inhibits TREK-1 currents by separate Ca 2+ - and ATP hydrolysis–dependent pathways (J.J. Enyeart et al, 2005, 2011; Liu et al, 2007). It will be interesting to determine whether AngII inhibits the noninactivating K + current in human cells by dual signaling pathways.…”