TRPC5 channel sensitivities to antioxidants and hydroxylated stilbenes.
TRPC5 channel sensitivities to antioxidants and hydroxylated stilbenes.
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TRPC5通道对抗氧化剂和羟基苯甲酸的敏感性。
DOI：
10.1074/jbc.m110.196956
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发表时间：
2011-02-18
期刊：
影响因子：
--
通讯作者：
Beech DJ
中科院分区：
文献类型：
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作者：
Naylor J;Al-Shawaf E;McKeown L;Manna PT;Porter KE;O'Regan D;Muraki K;Beech DJ
瞬时受体电位规范 5 (TRPC5) 形成阳离子通道，该通道是氧化磷脂、过氧化氢和还原硫氧还蛋白等因子的多模式传感器。本研究的目的是通过研究膳食抗氧化剂来扩展对 TRPC5 化学传感能力的了解。人 TRPC5 通道在 HEK 293 细胞中表达，并通过膜片钳和细胞内 Ca2+ 记录进行研究。 GFP 和 HA 标记的通道用于量化质膜定位。如果 TRPC5 活性是由外源性过氧化氢或镧系离子引起的，但不是由溶血磷脂酰胆碱或卡巴胆碱引起的，则没食子酸和维生素 C 会抑制 TRPC5 活性。过氧化氢酶模拟了这种效应，表明镧系元素诱发的活性依赖于内源性过氧化氢。相比之下，反式白藜芦醇抑制 TRPC5 的所有模式，并且其作用与维生素 C 的作用相加，表明其作用不依赖于抗氧化剂。 IC50 约为 10 μm。己烯雌酚是一种相关的羟基化芪，以相似的 IC50 抑制 TRPC5，但其作用与外膜斑块中白藜芦醇的作用形成鲜明对比，其中己烯雌酚引起强烈且可逆的抑制，而白藜芦醇没有作用，表明白藜芦醇的间接调节。白藜芦醇不影响通道表面密度，但其作用是钙敏感的，表明通过钙依赖性中间体发挥作用。这些数据表明，TRPC5 通过多种机制实现了以前未被认识的化学传感特性：（i）活性氧清除剂的抑制，因为 TRPC5 活性的一种模式依赖于内源性过氧化氢； (ii) 己烯雌酚直接阻断通道； (iii) 白藜芦醇的间接、不依赖于抗氧化剂的抑制作用。
Transient receptor potential canonical 5 (TRPC5) forms cationic channels that are polymodal sensors of factors including oxidized phospholipids, hydrogen peroxide, and reduced thioredoxin. The aim of this study was to expand knowledge of the chemical-sensing capabilities of TRPC5 by investigating dietary antioxidants. Human TRPC5 channels were expressed in HEK 293 cells and studied by patch clamp and intracellular Ca2+ recording. GFP- and HA-tagged channels were used to quantify plasma membrane localization. Gallic acid and vitamin C suppressed TRPC5 activity if it was evoked by exogenous hydrogen peroxide or lanthanide ions but not by lysophosphatidylcholine or carbachol. Catalase mimicked the effects, suggesting that lanthanide-evoked activity depended on endogenous hydrogen peroxide. Trans-resveratrol, by contrast, inhibited all modes of TRPC5, and its effect was additive with that of vitamin C, suggesting antioxidant-independent action. The IC50 was ∼10 μm. Diethylstilbestrol, a related hydroxylated stilbene, inhibited TRPC5 with a similar IC50, but its action contrasted sharply with that of resveratrol in outside-out membrane patches where diethylstilbestrol caused strong and reversible inhibition and resveratrol had no effect, suggesting indirect modulation by resveratrol. Resveratrol did not affect channel surface density, but its effect was calcium-sensitive, indicating an action via a calcium-dependent intermediate. The data suggest previously unrecognized chemical-sensing properties of TRPC5 through multiple mechanisms: (i) inhibition by scavengers of reactive oxygen species because a mode of TRPC5 activity depends on endogenous hydrogen peroxide; (ii) direct channel blockade by diethylstilbestrol; and (iii) indirect, antioxidant-independent inhibition by resveratrol.