Kang Mingmin, Zhang Yanming, Ding Xiufang, Xu Jianfu, Pang Xiaoyun
State key Laboratory of NBC Protection for Civilian, Beijing102205, China.
Key Laboratory of Biomacromolecules (Chinese Academy of Sciences), National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Membranes (Basel). 2025 Jun 19;15(6):187. doi: 10.3390/membranes15060187.
TRPA1 (Transient Receptor Potential Ankyrin 1), a cation channel predominantly expressed in sensory neurons, plays a critical role in detecting noxious stimuli and mediating pain signal transmission. As a key player in nociceptive signaling pathways, TRPA1 has emerged as a promising therapeutic target for the development of novel analgesics. Crotalphine (CRP), a 14-amino acid peptide, has been demonstrated to specifically activate TRPA1 and elicit potent analgesic effects. Previous cryo-EM (cryo-electron microscopy) studies have elucidated the structural mechanisms of TRPA1 activation by small-molecule agonists, such as iodoacetamide (IA), through covalent modification of N-terminal cysteine residues. However, the molecular interactions between TRPA1 and peptide ligands, including crotalphine, remain unclear. Here, we present the cryo-EM structure of ligand-free human TRPA1 consistent with the literature, as well as TRPA1 complexed with crotalphine, with resolutions of 3.1 Å and 3.8 Å, respectively. Through a combination of single-particle cryo-EM studies, patch-clamp electrophysiology, and microscale thermophoresis (MST), we have identified the cysteine residue at position 621 (Cys621) within the TRPA1 ion channel as the primary binding site for crotalphine. Upon binding to the reactive pocket containing C621, crotalphine induces rotational and translational movements of the transmembrane domain. This allosteric modulation coordinately dilates both the upper and lower gates, facilitating ion permeation.
瞬时受体电位锚蛋白1（TRPA1）是一种主要在感觉神经元中表达的阳离子通道，在检测有害刺激和介导疼痛信号传递中起关键作用。作为伤害性信号通路中的关键因子，TRPA1已成为开发新型镇痛药的一个有前景的治疗靶点。响尾蛇肽（CRP）是一种由14个氨基酸组成的肽，已被证明能特异性激活TRPA1并产生强效镇痛作用。先前的冷冻电镜（cryo-EM）研究已经阐明了小分子激动剂（如碘乙酰胺（IA））通过共价修饰N端半胱氨酸残基激活TRPA1的结构机制。然而，TRPA1与包括响尾蛇肽在内的肽配体之间的分子相互作用仍不清楚。在此，我们展示了与文献一致的无配体人TRPA1的冷冻电镜结构，以及与响尾蛇肽复合的TRPA1的冷冻电镜结构，分辨率分别为3.1 Å和3.8 Å。通过结合单颗粒冷冻电镜研究、膜片钳电生理学和微量热泳动（MST）技术，我们确定了TRPA1离子通道中621位的半胱氨酸残基（Cys621）是响尾蛇肽的主要结合位点。响尾蛇肽与含有C621的反应口袋结合后，会诱导跨膜结构域的旋转和平移运动。这种变构调节协同扩张上下门控，促进离子通透。
