摘要
Transient receptor potential vanilloid 1(TRPV1)channel plays an important role in a wide range of physiological and pathological processes,and a comprehensive understanding of TRPV1 gating will create opportunities for therapeutic intervention.Recent incredible advances in cryo-electron microscopy(cryo-EM)have yielded high-resolution structures of all TRPV subtypes(TRPV1-6)and all of them share highly conserved six transmembrane(TM)domains(S1-S6).As revealed by the open structures of TRPV1 in the presence of a bound vanilloid agonist(capsaicin or resiniferatoxin),TM helices S1 to S4 form a bundle that remains quiescent during channel activation,highlighting differences in the gating mechanism of TRPV1 and voltage-gated ion channels.Here,however,we argue that the structural dynamics rather than quiescence of S1-S4 domains is necessary for capsaicin-mediated activation of TRPV1.Using fluorescent unnatural amino acid(flUAA)incorporation and voltage-clamp fluorometry(VCF)analysis,we directly observed allostery of the S1-S4 bundle upon capsaicin binding.Covalent occupation of VCF-identified sites,single-channel recording,cell apoptosis analysis,and exploration of the role of PSFL828,a novel non-vanilloid agonist we identified,have collectively confirmed the essential role of this coordinated S1-S4 motility in capsaicin-mediated activation of TRPV1.This study concludes that,in contrast to cryo-EM structural studies,vanilloid agonists are also required for S1-S4 movement during TRPV1 activation.Redefining the gating process of vanilloid agonists and the discovery of new non-vanilloid agonists will allow the evaluation of new strategies aimed at the development of TRPV1 modulators.
瞬时受体电位香草素1(TRPV1)通道参与了哺乳动物体内许多重要的生理和病理过程,对其门控机制的全面了解有助于为相关疾病的治疗干预提供可能.近年来冷冻电子显微镜(cryo-EM)技术在结构生物学中取得了惊人的成果,所有TRPV亚型(TRPV1±6)的高分辨率结构也得到了解析:它们都具有高度保守的6个跨膜(TM)结构域(S1±S6),与电压门控离子通道(VGICs)相似.并且,在结合了香草素激动剂(辣椒素或树脂毒素)的TRPV1通道开放结构中,TM螺旋S1±S4形成一束,保持静止状态,这也证明了TRP和VGICs通道功能和门控机制的差异.然而,本研究认为S1±S4的动态变化而非静止是辣椒素激活TRPV1的必要条件.通过荧光非天然氨基酸结合和电压钳荧光测量分析,本研究直接观察到辣椒素结合后S1±S4域的运动.电压钳荧光测量法(VCF)所识别位点的化学修饰、单通道记录、细胞凋亡分析和对一种新激动剂PSFL828作用的探索,共同证实了这种协调的S1±S4运动在辣椒素介导的TRPV1激活中的重要作用.这项研究提出了一个新的见解:与冷冻电镜结构研究的结论不同,香草素激动剂在TRPV1激活过程中也需要S1±S4运动.本文重新定义香草素类激动剂的门控过程并发现了新的非香草素类激动剂,为开发TRPV1调节剂提供了新策略.
作者
Meng-Yang Sun
Xue Zhang
Peng-Cheng Yu
Di Liu
Yang Yang
Wen-Wen Cui
Xiao-Na Yang
Yun-Tao Lei
Xing-Hua Li
Wen-Hui Wang
Peng Cao
Heng-Shan Wang
Michael X.Zhu
Chang-Zhu Li
Rui Wang
Ying-Zhe Fan
Ye Yu
孙萌阳;张雪;于彭城;刘迪;杨扬;崔雯雯;杨晓娜;雷运涛;李兴华;王文辉;曹鹏;王恒山;朱曦;李昌珠;王锐;樊瀛哲;于烨(School of Life Sciences and Key Laboratory of Preclinical Study for New Drugs of Gansu Province,School of Basic Medical Sciences,Lanzhou University,Lanzhou 730000,China;State Key Laboratory of Utilization of Woody Oil Resource,Hunan Academy of Forestry,Changsha 410004,China;Department of Pharmacology and Chemical Biology,Institute of Medical Sciences,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Department of Basic Medicine and Clinical Pharmacy,China Pharmaceutical University,Nanjing 210009,China;Putuo Hospital,Shanghai University of Chinese Traditional Medicine,Shanghai 200062,China;College of Bioscience and Biotechnology,Hunan Agricultural University,Changsha 410128,China;Hospital of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210023,China;State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources,Collaborative Innovation Center for Guangxi Ethnic Medicine,School of Chemistry and Pharmaceutical Sciences,Guangxi Normal University,Guilin 541004,China;Department of Integrative Biology and Pharmacology,McGovern Medical School,The University of Texas Health Science Center at Houston,Houston,TX 77030,USA)
基金
supported by the Natural Science Foundation of Jiangsu Province(BK20202002)
the National Natural Science Foundation of China(81603409,31900808,81902480,21977021,31570832,31971146,and 31971042)
Innovation and Entrepreneurship Talent Program of Jiangsu Province
State Key Laboratory of Utilization of Woody Oil Resource(2019XK2002)
the Natural Science Foundation of Hunan Province(2018JJ1012)
Hunan“Huxiang”High-level Talent Program(2021)
“Xing Yao”Leading Scholars of China Pharmaceutical University(2021)。