Benzoquinone Reveals a Cysteine-Dependent Desensitization Mechanism of TRPA1
Benzoquinone Reveals a Cysteine-Dependent Desensitization Mechanism of TRPA1
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DOI：
10.1124/mol.112.084194
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发表时间：
2013-05-01
影响因子：
3.6
通讯作者：
Blair, Nathaniel T.
中科院分区：
文献类型：
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作者：
Ibarra, Yessenia;Blair, Nathaniel T.
瞬时受体电位锚蛋白 1 (TRPA1) 非选择性阳离子通道在后生动物的大部分区域具有作为有毒化学传感器的保守功能。亲电化学物质通过氨基末端区域半胱氨酸残基的共价修饰来激活昆虫和脊椎动物 TRPA1。尽管天然存在的亲电植物化合物（例如芥末油和肉桂醛）是 TRPA1 激动剂，但尚不清楚节肢动物产生的亲电子物质是否会激活哺乳动物 TRPA1。我们表征了亲电节肢动物防御化合物对苯醌 (pBQN) 对人类 TRPA1 通道的影响。我们使用异源表达野生型 TRPA1 或具有三个对亲电子激活至关重要的丝氨酸取代半胱氨酸（C621S、C641S、C665S）的 TRPA1 的人胚胎肾细胞的全细胞记录。我们发现 pBQN 从 10 nM 开始激活 TRPA1，在 300 nM 时达到峰值；较高的浓度导致快速激活，然后快速下降。 pBQN 的激活需要与半胱氨酸残基发生反应，但这些残基与之前报道的亲电子试剂的关键目标不同。我们发现在较高 pBQN 浓度下当前的减少是 TRPA1 的半胱氨酸依赖性脱敏，并且不需要事先激活。脱敏所需的半胱氨酸并非所有亲电子试剂都能接触到，因为碘乙酰胺和内部应用的 2-(三甲基铵)乙基甲磺酸盐未能引起脱敏（尽管有大量活化）。有趣的是，pBQN 脱敏后，野生型 TRPA1 对非亲电子激动剂香芹酚的反应显着降低，而三重半胱氨酸突变体 TRPA1 保留了其全部反应。我们的结果表明，亲电化合物对多个半胱氨酸残基的修饰可以产生 TRPA1 通道的激活和脱敏。
The transient receptor potential ankyrin 1 (TRPA1) nonselective cation channel has a conserved function as a noxious chemical sensor throughout much of Metazoa. Electrophilic chemicals activate both insect and vertebrate TRPA1 via covalent modification of cysteine residues in the amino-terminal region. Although naturally occurring electrophilic plant compounds, such as mustard oil and cinnamaldehyde, are TRPA1 agonists, it is unknown whether arthropod-produced electrophiles activate mammalian TRPA1. We characterized the effects of the electrophilic arthropod defensive compound para-benzoquinone (pBQN) on the human TRPA1 channel. We used whole-cell recordings of human embryonic kidney cells heterologously expressing either wild-type TRPA1 or TRPA1 with three serine-substituted cysteines crucial for electrophile activation (C621S, C641S, C665S). We found that pBQN activates TRPA1 starting at 10 nM and peaking at 300 nM; higher concentrations caused rapid activation followed by a fast decline. Activation by pBQN required reactivity with cysteine residues, but ones that are distinct from those previously reported to be the key targets of electrophiles. The current reduction we found at higher pBQN concentrations was a cysteine-dependent desensitization of TRPA1, and did not require prior activation. The cysteines required for desensitization are not accessible to all electrophiles as iodoacetamide and internally applied 2-(trimethylammonium) ethyl methanesulfonate failed to cause desensitization (despite large activation). Interestingly, following pBQN desensitization, wild-type TRPA1 had dramatically reduced response to the nonelectrophile agonist carvacrol, whereas the triple cysteine mutant TRPA1 retained its full response. Our results suggest that modification of multiple cysteine residues by electrophilic compounds can generate both activation and desensitization of the TRPA1 channel.