Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin.
Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin.
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DOI：
10.1152/ajpcell.00117.2011
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发表时间：
2011-05
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影响因子：
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通讯作者：
J. J. Enyeart-J.;Haiyan Liu;J. A. Enyeart
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作者：
J. J. Enyeart-J.;Haiyan Liu;J. A. Enyeart
牛肾上腺皮质细胞表达 bTREK-1 K(+)（牛 KCNK2）通道，该通道可通过单独的 Ca(2+) 和 ATP 水解依赖性信号通路通过 Gq 偶联受体被 ANG II 抑制。肾上腺束状带 (AZF) 细胞的全细胞和单膜片钳记录用于表征 bTREK-1 的 Ca(2+) 依赖性抑制。在使用含有 0.5 mM EGTA 但不含 ATP 的移液管溶液进行的全细胞记录中，Ca(2+) 离子载体离子霉素 (1 μM) 对 bTREK-1 产生短暂抑制，并在几分钟内自发逆转。在较高浓度下，离子霉素 (5-10 μM) 对 bTREK-1 产生持续抑制，即使在不存在可水解 [ATP](i) 的情况下，这种抑制在洗涤后也是可逆的。 BAPTA 在抑制 ANG II 对 bTREK-1 的抑制方面比 EGTA 更有效。当用 11 mM BAPTA 或 EGTA 将细胞内 Ca(2+) 浓度 ([Ca(2+)](i)) 缓冲至 20 nM 时，ANG II (10 nM) 分别抑制 bTREK-1 12.0 ± 4.5% (n=11) 和 59.3 ± 8.4% (n=4)。在移液管中加入水溶性磷脂酰肌醇 4,5-二磷酸 (PIP(2)) 类似物 DiC(8)PI(4,5)P(2) 未能增加 bTREK-1 表达或减少 ANG II 对其的抑制。 Ca(2+) (10-35 μM) 以浓度依赖性方式降低了从内向外斑片记录的单一 bTREK-1 通道的开放概率 (P(o))。这些结果与 ANG II 通过 Ca(2+) 依赖性机制抑制 bTREK-1 K(+) 通道的模型一致，该机制不需要消耗膜相关的 PIP(2)。他们进一步表明，Ca(2+) 源位于bTREK-1 通道的“Ca(2+) 纳米域”内，其中[Ca(2+)](i) 的浓度可能达到>10 μM。 bTREK-1 是第一个被证明可通过激活 G 蛋白偶联受体而被 Ca(2+) 抑制的双孔 K(+) 通道。
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 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 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 μM) 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 PIP(2). 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 μM. bTREK-1 is the first two-pore K(+) channel shown to be inhibited by Ca(2+) through activation of a G protein-coupled receptor.