title: Intersubunit physical couplings fostered by the left flipper domain facilitate channel opening of P2X4 receptors.
abstract: P2X receptors are ATP-gated trimeric channels with important roles in diverse pathophysiological functions. A detailed understanding of the mechanism underlying the gating process of these receptors is thus fundamentally important and may open new therapeutic avenues. The left flipper (LF) domain of the P2X receptors is a flexible loop structure, and its coordinated motions together with the dorsal fin (DF) domain are crucial for the channel gating of the P2X receptors. However, the mechanism underlying the crucial role of the LF domain in the channel gating remains obscure. Here, we propose that the ATP-induced allosteric changes of the LF domain enable it to foster intersubunit physical couplings among the DF and two lower body domains, which are pivotal for the channel gating of P2X4 receptors. Metadynamics analysis indicated that these newly established intersubunit couplings correlate well with the ATP-bound open state of the receptors. Moreover, weakening or strengthening these physical interactions with engineered intersubunit metal bridges remarkably decreased or increased the open probability of the receptors, respectively. Further disulfide cross-linking and covalent modification confirmed that the intersubunit physical couplings among the DF and two lower body domains fostered by the LF domain at the open state act as an integrated structural element that is stringently required for the channel gating of P2X4 receptors. Our observations provide new mechanistic insights into P2X receptor activation and will stimulate development of new allosteric modulators of P2X receptors.
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Liang-Fei
authors.initials: LF
authors.lastname: Sun
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Wen-Wen
authors.initials: WW
authors.lastname: Cui
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Wen-Shan
authors.initials: WS
authors.lastname: Zhao
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.|the College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China, and.
authors.forename: Xue-Fei
authors.initials: XF
authors.lastname: Ma
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.|the College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China, and.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: You-Min
authors.initials: YM
authors.lastname: Hu
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Li-Dong
authors.initials: LD
authors.lastname: Huang
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Xiao-Yang
authors.initials: XY
authors.lastname: Cheng
authors.affiliation: the Department of Anesthesiology and Perioperative Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
authors.forename: Lingyong
authors.initials: L
authors.lastname: Li
authors.affiliation: the College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China, and.
authors.forename: Xiang-Yang
authors.initials: XY
authors.lastname: Lu
authors.affiliation: the College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China, and tianyun79616@163.com.
authors.forename: Yun
authors.initials: Y
authors.lastname: Tian
authors.affiliation: From the Department of Pharmacology and Chemical Biology, Institute of Medical Sciences and Hongqiao International Institute of Medicine of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China, yuye@shsmu.edu.cn.|the College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China, and.
authors.forename: Ye
authors.initials: Y
authors.lastname: Yu
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D056004:Molecular Dynamics Simulation; D000072417:Protein Domains; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D058848:Receptors, Purinergic P2X4
keywords: P2X receptors; conformational change; gating; ion channel; molecular simulations; physical couplings; protein domain; receptor structure-function; single channel recording