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

Enyeart JJ, Liu H, Enyeart JA. Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin. Am J Physiol Cell Physiol 301: C619-C629, 2011. First published May 25, 2011; doi: 10.1152/ajpcell.00117.2011.-Bovine adrenocortical cells express bTREK-1 K(+) (bovine KCNK2) channels that are inhibited by ANG II through a Gq-coupled receptor by separate Ca(2+) and ATP hydrolysis-dependent signaling pathways. Whole cell and single patch clamp recording from adrenal zona fasciculata (AZF) cells were used to characterize Ca(2+)-dependent inhibition of bTREK-1. In whole cell recordings with pipette solutions containing 0.5 mM EGTA and no ATP, the Ca(2+) ionophore ionomycin (1 mu M) produced a transient inhibition of bTREK-1 that reversed spontaneously within minutes. At higher concentrations, ionomycin (5-10 mu 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(2+) concentration ([Ca(2+)](i)) was buffered to 20 nM with either 11 mM BAPTA or EGTA, ANG II (10 nM) inhibited bTREK-1 by 12.0 +/- 4.5% (n = 11) and 59.3 +/- 8.4% (n = 4), respectively. Inclusion of the water-soluble phosphatidylinositol 4,5-bisphosphate (PIP(2)) analog DiC(8)PI(4,5)P(2) 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(2+) (10-35 mu M) in a concentration-dependent manner. These results are consistent with a model in which ANG II inhibits bTREK-1 K(+) channels by a Ca(2+)-dependent mechanism that does not require the depletion of membrane-associated PIP2. They further indicate that the Ca(2+) source is located in close proximity within a Ca(2+) nanodomain of bTREK-1 channels, where [Ca(2+)](i) may reach concentrations of > 10 mu M. bTREK-1 is the first two-pore K(+) channel shown to be inhibited by Ca(2+) through activation of a G protein-coupled receptor.