Department of Physiology and Biophysics, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA.
Pflugers Arch. 2012 Nov;464(5):471-80. doi: 10.1007/s00424-012-1159-y. Epub 2012 Sep 25.
Reactive oxygen species (ROS) generated by mitochondria or NADPH oxidase have been implicated in the inhibition of K(+) current by hypoxia in chemoreceptor cells. As TASKs are highly active background K(+) channels in these cells, we studied the role of ROS in hypoxia-induced inhibition of TASKs. In HeLa cells expressing TASKs, H(2)O(2) applied to inside-out patches activated TASK-1, TASK-3, and TASK-1/3 heteromer starting at ~16 mM. When applied to cell-attached or outside-out patches, 326 mM H(2)O(2) did not affect TASK activity. Other K(2P) channels (TREK-1, TREK-2, TASK-2, TALK-1, TRESK) were not affected by H(2)O(2) (tested up to 326 mM). A reducing agent (dithiothreitol) and a cysteine-modifying agent (2-aminoethyl methanethiosulfonate hydrobromide) had no effect on basal TASK activity and did not block the H(2)O(2)-induced increase in channel activity. A TASK mutant in which the C-terminus of TASK-3 was replaced with that of TREK-2 showed a normal sensitivity to H(2)O(2). Xanthine/xanthine oxidase mixture used to generate superoxide radical showed no effect on TASK-1, TASK-3, and TASK-1/3 heteromer from either side of the membrane, but it strongly activated TASK-2 from the extracellular side. Acute H(2)O(2) (32-326 mM) exposure did not affect hSlo1/b1(BK) expressed in HeLa cells and BK in carotid body glomus cells. In carotid body glomus cells, adrenal cortical cells, and cerebellar granule neurons that show abundant hypoxia-sensitive TASK activity, H(2)O(2) (>16 mM) activated the channels only when applied intracellularly, similar to that observed with cloned TASKs. These findings show that ROS do not support or inhibit TASK and BK activity and therefore are unlikely to be the hypoxic signal that causes cell excitation via inhibition of these K(+) channels.
活性氧（ROS）由线粒体或 NADPH 氧化酶产生，与化学感受器细胞缺氧时 K(+)电流的抑制有关。由于 TASK 是这些细胞中高度活跃的背景 K(+)通道，我们研究了 ROS 在缺氧诱导的 TASK 抑制中的作用。在表达 TASK 的 HeLa 细胞中，用 H(2)O(2)处理胞内面向的膜片钳激活 TASK-1、TASK-3 和 TASK-1/3 异源二聚体，起始于约 16 mM。当应用于细胞贴附或胞外面向的膜片钳时，326 mM H(2)O(2)对 TASK 活性没有影响。其他 K(2P)通道（TREK-1、TREK-2、TASK-2、TALK-1、TRESK）不受 H(2)O(2)的影响（测试高达 326 mM）。一种还原剂（二硫苏糖醇）和一种半胱氨酸修饰剂（2-氨基乙基甲硫基磺酸盐氢溴化物）对基础 TASK 活性没有影响，也不能阻断 H(2)O(2)诱导的通道活性增加。用 TREK-2 的 C 末端替换 TASK-3 的 C 末端的 TASK 突变体对 H(2)O(2)表现出正常的敏感性。用于生成超氧自由基的黄嘌呤/黄嘌呤氧化酶混合物对膜两侧的 TASK-1、TASK-3 和 TASK-1/3 异源二聚体均无影响，但它强烈激活胞外侧的 TASK-2。急性 H(2)O(2)（32-326 mM）暴露对 HeLa 细胞中表达的 hSlo1/b1(BK)和颈动脉体小球细胞中的 BK 没有影响。在颈动脉体小球细胞、肾上腺皮质细胞和小脑颗粒神经元中，大量存在缺氧敏感的 TASK 活性，当用 H(2)O(2)（>16 mM）处理胞内时，这些通道才被激活，类似于克隆 TASK 观察到的情况。这些发现表明，ROS 既不支持也不抑制 TASK 和 BK 活性，因此不太可能是通过抑制这些 K(+)通道导致细胞兴奋的缺氧信号。
