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

Abstract

Bovine adrenocortical cells express bTREK-1 K+(bovine KCNK2) channels that are inhibited by ANG II through a Gq-coupled receptor by separate Ca2+and ATP hydrolysis-dependent signaling pathways. Whole cell and single patch clamp recording from adrenal zona fasciculata (AZF) cells were used to characterize Ca2+-dependent inhibition of bTREK-1. In whole cell recordings with pipette solutions containing 0.5 mM EGTA and no ATP, the Ca2+ionophore ionomycin (1 μM) produced a transient inhibition of bTREK-1 that reversed spontaneously within minutes. At higher concentrations, ionomycin (5–10 μM) 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 Ca2+concentration ([Ca2+]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 (PIP2) analog DiC8PI(4,5)P2in the pipette failed to increase bTREK-1 expression or reduce its inhibition by ANG II. The open probability ( Po) of unitary bTREK-1 channels recorded from inside-out patches was reduced by Ca2+(10–35 μM) in a concentration-dependent manner. These results are consistent with a model in which ANG II inhibits bTREK-1 K+channels by a Ca2+-dependent mechanism that does not require the depletion of membrane-associated PIP2. They further indicate that the Ca2+source is located in close proximity within a “Ca2+nanodomain” of bTREK-1 channels, where [Ca2+]imay reach concentrations of >10 μM. bTREK-1 is the first two-pore K+channel shown to be inhibited by Ca2+through activation of a G protein-coupled receptor.