The Crucial Roles Of The Kinked Conformation Of ?2,3 Stablized By Salt Bridge In Channel Gating And Channelopathies Of P2X Receptors

P2X receptors are non-selective ion channels gated by extracellular ATP.The channels are non-selectively permeable to cations,such as Na⁺,K⁺ and Ca²⁺,through binding of extracellular ATP.Seven P2X have been cloned from eukaryotic cells,denoting P2X1,P2X2,P2X3,P2X4,P2X5,P2X6 and P2X7,which can form homomeric or heteromeric trimers.P2X receptors are widely expressed in all mammalian tissues,involved in a large array of physiological processes such as cardiovascular system,nervous system,respiratory system,gastrointestinal system,and the urogenital system.Dysfunctions of P2X are associated with several diseases,including multiple sclerosis,rheumatoid arthritis,chronic neuropathic pain,stroke,thrombosis and so on.As new targets for drug design,more than 20 small molecule compounds targeted P2X receptors in the therapy of rheumatoid arthritis,thrombosis,kinds of pain syndrome have entered pre-clinical or phase II or III clinical trials.Very recently,the resolved crystal structures of zfP2X4 at open and resting states have provided us the structural basis to investigate the allosteric changes in channel gating.In the post-structure era,these structures advanced our understanding of the allosteric conformational changes of this ion channel at the atomic level.Significant progresses in determining the essential role of residues/motifs in channel activation of P2X receptors have been made in the pre-structure era.Recently,the structural determination of P2X receptors provided us an opportunity to make a fresh appraisal on the role of those residues/motifs.Resides R309 and D85?rat P2X4 numbering?are highly conserved throughout the P2X family and were involved in loss-of-function polymorphism in the human P2X receptors.Previous studies implied their roles in ATP-binding,however,the crystal structures demonstrate that those two residues form a salt-bridge and locate far away from ATP-binding site?the shortest atomic distance between ATP and the residue of the salt bridge is over 12 A?which is too far to produce direct interactions between the residues and the ATP.Thus,the role of this salt-bridge should be reevaluated.Here,we demonstrated the crucial role of this structural element both in protein expression and channel gating rather than ATP-binding.A combination using of mutagenesis,charge swap and disulfide crosslinking revealed the stringentrequirement of this salt-bridge both in protein expression and channel activation.This salt-bridge may contribute to stabilize the bending conformation of ?2,3-sheet,a structure elements structurally coupled towards a2 helix.Strongly kinked ?2,3 is essential for the domain-domain interactions between head,dorsal fin,right flipper domains,and loop ?7,8 in P2X4 receptors.Disulfide crosslinking with directions inversing or along the bending angle of(32,3-sheet towards a2 led to loss-and gain-of-function of P2X4 receptors,respectively.Further insertion of amino acids with bulky side chains in linker between ?2,3-sheet and the alteration in the conformation of ?2 to change the kinked conformation of ?2,3 led to loss-of-function of P2X4 receptors.All these findings provided us a renewed understanding of the role of salt bridge D85...R309 and its structurally coupled(32,3-sheet in the functional P2X receptors.Here,we uncover an intermediated state indispensable for channel activation of P2X4 receptors.As revealed by metadynamics,minimum energy pathway,molecular dynamic simulations,an intermediated state occurred during channel gating of P2X4 receptors.Mutagenesis,engineered disulfide crosslinking,metal bridge and covalent modification to prevent the occurring of this intermediated state,lead to impair or full abolished channel activation of P2X4 receptors,confirming the existence and essential role of this intermediated sate.Disulfide crosslinking to curb the relative motions between beak domain and upper body domain significantly influenced agonist-recognition and channel gating of P2X4 receptors,suggesting a crucial role of this intermediated state in accommodating ATP and coordinating the conformational changes of those key domains during channel gating.Thus,the intermediated conformational transition identified here is indispensable for the relative motions between beak domain and upper body domain during channel gating of P2X4 receptors.Our observation provides novel mechanistic insight into P2X activation and contributes to develop new strategies for designing new drugs targeting on those unique trimetric ligand-gated ion channels.