Macpherson Lindsey J, Dubin Adrienne E, Evans Michael J, Marr Felix, Schultz Peter G, Cravatt Benjamin F, Patapoutian Ardem
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Nature. 2007 Feb 1;445(7127):541-5. doi: 10.1038/nature05544. Epub 2007 Jan 21.
The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.
神经系统通过传递疼痛信号的伤害性神经元感知外周损伤。TRPA1是瞬时受体电位(TRP)离子通道家族的成员,在伤害性神经元中表达。TRPA1可被多种有害刺激激活,包括低温、刺激性天然化合物和环境刺激物。目前尚不清楚这些多样的刺激如何激活TRPA1。我们观察到,大多数已知能激活TRPA1的化合物都能够与半胱氨酸残基共价结合。在此,我们使用点击化学方法表明,两种此类化合物——芥子油和肉桂醛的衍生物能够与小鼠TRPA1共价结合。结构不相关的半胱氨酸修饰剂,如碘乙酰胺(IA)和(2-氨基乙基)甲硫基磺酸盐(MTSEA),也能结合并激活TRPA1。我们通过质谱鉴定出14个被IA标记的胞质TRPA1半胱氨酸,其中3个是正常通道功能所必需的。在切除的膜片中,活性化合物激活了TRPA1电流,在无钙溶液中洗去化合物后,该电流至少维持10分钟。最后,形成二硫键的MTSEA对TRPA1的激活被还原剂二硫苏糖醇(DTT)阻断。总的来说,我们的数据表明,TRPA1内反应性半胱氨酸的共价修饰可导致通道激活,通过疼痛途径快速发出潜在组织损伤的信号。