title: A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of β2,3-Sheet Essential for Channel Function of P2X4 Receptors.
abstract: Significant progress has been made in understanding the roles of crucial residues/motifs in the channel function of P2X receptors during the pre-structure era. The recent structural determination of P2X receptors allows us to reevaluate the role of those residues/motifs. Residues Arg-309 and Asp-85 (rat P2X4 numbering) are highly conserved throughout the P2X family and were involved in loss-of-function polymorphism in human P2X receptors. Previous studies proposed that they participated in direct ATP binding. However, the crystal structure of P2X demonstrated that those two residues form an intersubunit salt bridge located far away from the ATP-binding site. Therefore, it is necessary to reevaluate the role of this salt bridge in P2X receptors. Here, we suggest the crucial role of this structural element both in protein stability and in channel gating rather than direct ATP interaction and channel assembly. Combining mutagenesis, charge swap, and disulfide cross-linking, we revealed the stringent requirement of this salt bridge in normal P2X4 channel function. This salt bridge may contribute to stabilizing the bending conformation of the β2,3-sheet that is structurally coupled with this salt bridge and the α2-helix. Strongly kinked β2,3 is essential for domain-domain interactions between head domain, dorsal fin domain, right flipper domain, and loop β7,8 in P2X4 receptors. Disulfide cross-linking with directions opposing or along the bending angle of the β2,3-sheet toward the α2-helix led to loss-of-function and gain-of-function of P2X4 receptors, respectively. Further insertion of amino acids with bulky side chains into the linker between the β2,3-sheet or the conformational change of the α2-helix, interfering with the kinked conformation of β2,3, led to loss-of-function of P2X4 receptors. All these findings provided new insights in understanding the contribution of the salt bridge between Asp-85 and Arg-309 and its structurally coupled β2,3-sheet to the function of P2X receptors.
authors: From the 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, the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: Wen-Shan
authors: WS
authors: Zhao
authors: From the 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, the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: Meng-Yang
authors: MY
authors: Sun
authors: the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: Liang-Fei
authors: LF
authors: Sun
authors: the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: Yan
authors: Y
authors: Liu
authors: the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: Yang
authors: Y
authors: Yang
authors: the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: Li-Dong
authors: LD
authors: Huang
authors: the Putuo District Center Hospital, Shanghai University of Chinese Traditional Medicine, Shanghai 200062, China.
authors: Ying-Zhe
authors: YZ
authors: Fan
authors: the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: Xiao-Yang
authors: XY
authors: Cheng
authors: the Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China, and the Laboratory of Cellular and Molecular Biology, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, China.
authors: Peng
authors: P
authors: Cao
authors: the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: You-Min
authors: YM
authors: Hu
authors: the Department of Anesthesiology and Perioperative Medicine, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030.
authors: Lingyong
authors: L
authors: Li
authors: the College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China.
authors: Yun
authors: Y
authors: Tian
authors: From the 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, wangrui@lzu.edu.cn.
authors: Rui
authors: R
authors: Wang
authors: the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China, the College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China, yuye@shsmu.edu.cn.
authors: Ye
authors: Y
authors: Yu
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry* / Q000378:metabolism*; D012492:Salts / Q000737:chemistry; D016415:Sequence Alignment
chemical_list: D004220:Disulfides; D058848:Receptors, Purinergic P2X4; D012492:Salts; D000255:Adenosine Triphosphate
keywords: ATP; P2X receptors; conformational change; ion channel; protein expression; protein stability; salt bridge