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Intersubunit physical couplings fostered by the left flipper domain facilitate channel opening of P2X4 receptors

  • Jin Wang
    Affiliations
    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,
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  • Liang-Fei Sun
    Affiliations
    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,
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  • Wen-Wen Cui
    Affiliations
    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,
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  • Wen-Shan Zhao
    Affiliations
    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,
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  • Xue-Fei Ma
    Affiliations
    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
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  • Bin Li
    Affiliations
    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
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  • Yan Liu
    Affiliations
    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,
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  • Yang Yang
    Affiliations
    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,
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  • You-Min Hu
    Affiliations
    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,
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  • Li-Dong Huang
    Affiliations
    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,
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  • Xiao-Yang Cheng
    Affiliations
    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,
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  • Lingyong Li
    Affiliations
    the Department of Anesthesiology and Perioperative Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
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  • Xiang-Yang Lu
    Affiliations
    the College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China, and
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  • Yun Tian
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    the College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China, and
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  • Ye Yu
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    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
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Open AccessPublished:March 16, 2017DOI:https://doi.org/10.1074/jbc.M116.771121
      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.

      Introduction

      P2X receptors are trimeric membrane ion channels (
      • Baconguis I.
      • Hattori M.
      • Gouaux E.
      Unanticipated parallels in architecture and mechanism between ATP-gated P2X receptors and acid sensing ion channels.
      ,
      • Kellenberger S.
      • Grutter T.
      Architectural and functional similarities between trimeric ATP-gated P2X receptors and acid-sensing ion channels.
      ) activated by extracellular ATP (
      • Coddou C.
      • Yan Z.
      • Obsil T.
      • Huidobro-Toro J.P.
      • Stojilkovic S.S.
      Activation and regulation of purinergic P2X receptor channels.
      ). So far, seven P2X subtypes (P2X1–P2X7) have been identified, which are expressed virtually in almost all mammalian tissues, including nervous, immune, and cardiovascular systems (
      • Coddou C.
      • Yan Z.
      • Obsil T.
      • Huidobro-Toro J.P.
      • Stojilkovic S.S.
      Activation and regulation of purinergic P2X receptor channels.
      ,
      • Khakh B.S.
      • North R.A.
      P2X receptors as cell-surface ATP sensors in health and disease.
      • Surprenant A.
      • North R.A.
      Signaling at purinergic P2X receptors.
      ). P2X receptors possess a molecular architecture distinct from other ion channel protein families (
      • Khakh B.S.
      • North R.A.
      P2X receptors as cell-surface ATP sensors in health and disease.
      ,
      • Jarvis M.F.
      • Khakh B.S.
      ATP-gated P2X cation-channels.
      ,
      • Kawate T.
      • Michel J.C.
      • Birdsong W.T.
      • Gouaux E.
      Crystal structure of the ATP-gated P2X(4) ion channel in the closed state.
      ) and are implicated in a wide range of physiological and pathological processes (
      • Kellenberger S.
      • Grutter T.
      Architectural and functional similarities between trimeric ATP-gated P2X receptors and acid-sensing ion channels.
      ,
      • Khakh B.S.
      • North R.A.
      P2X receptors as cell-surface ATP sensors in health and disease.
      ,
      • Surprenant A.
      • North R.A.
      Signaling at purinergic P2X receptors.
      ,
      • Burnstock G.
      • Nistri A.
      • Khakh B.S.
      • Giniatullin R.
      ATP-gated P2X receptors in health and disease.
      ), such as neuroinflammation, synaptic transmission, primary afferent signaling, chronic pain, central control of respiration, vascular remodeling, and the regulation of blood pressure. Accordingly, P2X receptors hold great interest as new therapeutic targets for inflammation and v cardiovascular and neurological diseases (
      • Burnstock G.
      • Nistri A.
      • Khakh B.S.
      • Giniatullin R.
      ATP-gated P2X receptors in health and disease.
      • Hattori M.
      • Gouaux E.
      Molecular mechanism of ATP binding and ion channel activation in P2X receptors.
      ,
      • North R.A.
      • Jarvis M.F.
      P2X receptors as drug targets.
      ,
      • North R.A.
      P2X receptors.
      ,
      • Burnstock G.
      Short- and long-term (trophic) purinergic signalling.
      ,
      • Kuan Y.H.
      • Shyu B.C.
      Nociceptive transmission and modulation via P2X receptors in central pain syndrome.
      • Beamer E.
      • Gölöncsér F.
      • Horváth G.
      • Bekő K.
      • Otrokocsi L.
      • Koványi B.
      • Sperlágh B.
      Purinergic mechanisms in neuroinflammation: an update from molecules to behavior.
      ). For this purpose, it is essential to fully understand the detailed gating mechanism of P2X receptors at the atomic level (
      • Hattori M.
      • Gouaux E.
      Molecular mechanism of ATP binding and ion channel activation in P2X receptors.
      ,
      • Jiang R.
      • Taly A.
      • Grutter T.
      Moving through the gate in ATP-activated P2X receptors.
      ,
      • Habermacher C.
      • Dunning K.
      • Chataigneau T.
      • Grutter T.
      Molecular structure and function of P2X receptors.
      ).
      High resolution X-ray structures at apo/closed and ATP-bound open states are available for zebrafish P2X4 (zfP2X4) (
      • Kawate T.
      • Michel J.C.
      • Birdsong W.T.
      • Gouaux E.
      Crystal structure of the ATP-gated P2X(4) ion channel in the closed state.
      ,
      • Hattori M.
      • Gouaux E.
      Molecular mechanism of ATP binding and ion channel activation in P2X receptors.
      ), human P2X3 (hP2X3) (
      • Mansoor S.E.
      • Lü W.
      • Oosterheert W.
      • Shekhar M.
      • Tajkhorshid E.
      • Gouaux E.
      X-ray structures define human P2X3 receptor gating cycle and antagonist action.
      ), Amblyomma maculatum P2X (AmP2X) (
      • Kasuya G.
      • Fujiwara Y.
      • Takemoto M.
      • Dohmae N.
      • Nakada-Nakura Y.
      • Ishitani R.
      • Hattori M.
      • Nureki O.
      Structural insights into divalent cation modulations of ATP-gated P2X receptor channels.
      ), and panda P2X7 (
      • Karasawa A.
      • Kawate T.
      Structural basis for subtype-specific inhibition of the P2X7 receptor.
      ) receptors, which greatly aid our understanding of the working principles of those unique receptors, such as the ligand recognition, pore architecture, and conformational changes associated with the channel activation. Based on these structures, optical control of P2X receptors independent of natural stimulus has been achieved via powerful optogating approaches (
      • Lemoine D.
      • Habermacher C.
      • Martz A.
      • Méry P.F.
      • Bouquier N.
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      Optical control of an ion channel gate.
      ,
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      Optical control of trimeric P2X receptors and acid-sensing ion channels.
      • Habermacher C.
      • Martz A.
      • Calimet N.
      • Lemoine D.
      • Peverini L.
      • Specht A.
      • Cecchini M.
      • Grutter T.
      Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel.
      ). The structural comparison of the closed and open structures suggests a possible gating mechanism of P2X receptors (Fig. 1, A and B) (
      • Hattori M.
      • Gouaux E.
      Molecular mechanism of ATP binding and ion channel activation in P2X receptors.
      ,
      • Habermacher C.
      • Dunning K.
      • Chataigneau T.
      • Grutter T.
      Molecular structure and function of P2X receptors.
      ,
      • Chataigneau T.
      • Lemoine D.
      • Grutter T.
      Exploring the ATP-binding site of P2X receptors.
      ,
      • Wang J.
      • Yu Y.
      Insights into the channel gating of P2X receptors from structures, dynamics and small molecules.
      ). First, at the ATP-binding site, ATP promotes the jaw closure between the head and dorsal fin (DF)
      The abbreviations used are: DF
      dorsal fin
      LF
      left flipper
      TM
      transmembrane
      PDB
      Protein Data Bank
      IVM
      ivermectin
      pF
      picofarad
      NPM
      N-phenylmaleimide
      MTSEA
      2-aminoethyl methanethiosulfonate
      MTSES
      2-sulfonatoethyl methanethiosulfonate
      DTNB
      5′-dithiobis(2-nitrobenzoic acid)
      ANOVA
      analysis of variance
      MD
      molecular dynamics
      CV
      collective variable
      pS
      picosiemens
      β-ME
      β-mercaptoethanol
      CHX
      cycloheximide.
      domains, making the DF domain move upward to the head domain to accommodate ATP. Meanwhile, the bound ATP pushes the left flipper (LF) domain out of the ATP-binding site. Second, because both the LF and DF domains are structurally coupled with the lower body domain, the movements of those two domains lead to a concomitant outward flexing of lower body domains in the open state, which markedly expands the central vestibule (Fig. 1B). Finally, lower body domains are directly coupled with two transmembrane (TM) domains TM1 and TM2, and therefore their outward flexing can directly promote the opening of ion channel pore by causing the TM helices to expand in an iris-like motion (Fig. 1B). Recent studies have partially confirmed this hypothesis (
      • Habermacher C.
      • Dunning K.
      • Chataigneau T.
      • Grutter T.
      Molecular structure and function of P2X receptors.
      ,
      • Chataigneau T.
      • Lemoine D.
      • Grutter T.
      Exploring the ATP-binding site of P2X receptors.
      ). Multiple approaches, including voltage-clamp fluorometry (
      • Lörinczi É.
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      • Taly A.
      • Kaczmarek-Hájek K.
      • Barrantes-Freer A.
      • Dutertre S.
      • Grutter T.
      • Rettinger J.
      • Nicke A.
      Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor.
      ,
      • Fryatt A.G.
      • Evans R.J.
      Kinetics of conformational changes revealed by voltage-clamp fluorometry give insight to desensitization at ATP-gated human P2X1 receptors.
      ), fast-scanning atomic force microscopy (
      • Shinozaki Y.
      • Sumitomo K.
      • Tsuda M.
      • Koizumi S.
      • Inoue K.
      • Torimitsu K.
      Direct observation of ATP-induced conformational changes in single P2X(4) receptors.
      ), electron microscopy (
      • Roberts J.A.
      • Allsopp R.C.
      • El Ajouz S.
      • Vial C.
      • Schmid R.
      • Young M.T.
      • Evans R.J.
      Agonist binding evokes extensive conformational changes in the extracellular domain of the ATP-gated human P2X1 receptor ion channel.
      ), engineering metal bridge (
      • Li M.
      • Kawate T.
      • Silberberg S.D.
      • Swartz K.J.
      Pore-opening mechanism in trimeric P2X receptor channels.
      • Jiang R.
      • Taly A.
      • Lemoine D.
      • Martz A.
      • Cunrath O.
      • Grutter T.
      Tightening of the ATP-binding sites induces the opening of P2X receptor channels.
      ,
      • Heymann G.
      • Dai J.
      • Li M.
      • Silberberg S.D.
      • Zhou H.X.
      • Swartz K.J.
      Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels.
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ), substituted cysteine accessibility (
      • Samways D.S.
      • Khakh B.S.
      • Dutertre S.
      • Egan T.M.
      Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors).
      ), normal mode analysis, and molecular dynamics (MD) simulations (
      • Jiang R.
      • Taly A.
      • Lemoine D.
      • Martz A.
      • Cunrath O.
      • Grutter T.
      Tightening of the ATP-binding sites induces the opening of P2X receptor channels.
      ,
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ,
      • Du J.
      • Dong H.
      • Zhou H.X.
      Gating mechanism of a P2X4 receptor developed from normal mode analysis and molecular dynamics simulations.
      • Huang L.D.
      • Fan Y.Z.
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      • Yang Y.
      • Liu Y.
      • Wang J.
      • Zhao W.S.
      • Zhou W.C.
      • Cheng X.Y.
      • Cao P.
      • Lu X.Y.
      • Yu Y.
      Inherent dynamics of head domain correlates with ATP-recognition of P2X4 receptors: insights gained from molecular simulations.
      ,
      • Kowalski M.
      • Hausmann R.
      • Dopychai A.
      • Grohmann M.
      • Franke H.
      • Nieber K.
      • Schmalzing G.
      • Illes P.
      • Riedel T.
      Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening.
      • Zhao W.S.
      • Sun M.Y.
      • Sun L.F.
      • Liu Y.
      • Yang Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Cao P.
      • Hu Y.M.
      • Li L.
      • Tian Y.
      • Wang R.
      • Yu Y.
      A highly conserved salt bridge stabilizes the kinked conformation of β2,3-sheet essential for channel function of P2X4 receptors.
      ), have demonstrated that the dynamics of the head domain (
      • Lörinczi É.
      • Bhargava Y.
      • Marino S.F.
      • Taly A.
      • Kaczmarek-Hájek K.
      • Barrantes-Freer A.
      • Dutertre S.
      • Grutter T.
      • Rettinger J.
      • Nicke A.
      Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor.
      ,
      • Shinozaki Y.
      • Sumitomo K.
      • Tsuda M.
      • Koizumi S.
      • Inoue K.
      • Torimitsu K.
      Direct observation of ATP-induced conformational changes in single P2X(4) receptors.
      ,
      • Roberts J.A.
      • Allsopp R.C.
      • El Ajouz S.
      • Vial C.
      • Schmid R.
      • Young M.T.
      • Evans R.J.
      Agonist binding evokes extensive conformational changes in the extracellular domain of the ATP-gated human P2X1 receptor ion channel.
      ,
      • Jiang R.
      • Taly A.
      • Lemoine D.
      • Martz A.
      • Cunrath O.
      • Grutter T.
      Tightening of the ATP-binding sites induces the opening of P2X receptor channels.
      ,
      • Huang L.D.
      • Fan Y.Z.
      • Tian Y.
      • Yang Y.
      • Liu Y.
      • Wang J.
      • Zhao W.S.
      • Zhou W.C.
      • Cheng X.Y.
      • Cao P.
      • Lu X.Y.
      • Yu Y.
      Inherent dynamics of head domain correlates with ATP-recognition of P2X4 receptors: insights gained from molecular simulations.
      ), tightening the ATP-binding site jaw (
      • Jiang R.
      • Taly A.
      • Lemoine D.
      • Martz A.
      • Cunrath O.
      • Grutter T.
      Tightening of the ATP-binding sites induces the opening of P2X receptor channels.
      ,
      • Huang L.D.
      • Fan Y.Z.
      • Tian Y.
      • Yang Y.
      • Liu Y.
      • Wang J.
      • Zhao W.S.
      • Zhou W.C.
      • Cheng X.Y.
      • Cao P.
      • Lu X.Y.
      • Yu Y.
      Inherent dynamics of head domain correlates with ATP-recognition of P2X4 receptors: insights gained from molecular simulations.
      ), and the expansion of upward (
      • Shinozaki Y.
      • Sumitomo K.
      • Tsuda M.
      • Koizumi S.
      • Inoue K.
      • Torimitsu K.
      Direct observation of ATP-induced conformational changes in single P2X(4) receptors.
      ) and central (
      • Samways D.S.
      • Khakh B.S.
      • Dutertre S.
      • Egan T.M.
      Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors).
      ,
      • Rokic M.B.
      • Stojilkovic S.S.
      • Zemkova H.
      Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating.
      ) vestibules are essential for the ATP recognition, ion permeation, channel activation, desensitization, and sustained activation of P2X receptors. However, more pronounced allosteric changes of the extracellular domain (
      • Fryatt A.G.
      • Evans R.J.
      Kinetics of conformational changes revealed by voltage-clamp fluorometry give insight to desensitization at ATP-gated human P2X1 receptors.
      ,
      • Shinozaki Y.
      • Sumitomo K.
      • Tsuda M.
      • Koizumi S.
      • Inoue K.
      • Torimitsu K.
      Direct observation of ATP-induced conformational changes in single P2X(4) receptors.
      • Roberts J.A.
      • Allsopp R.C.
      • El Ajouz S.
      • Vial C.
      • Schmid R.
      • Young M.T.
      • Evans R.J.
      Agonist binding evokes extensive conformational changes in the extracellular domain of the ATP-gated human P2X1 receptor ion channel.
      ) were observed in biochemical, biophysical, and computational analysis than those observed in the open structure of the P2X4 receptor. Recent studies (
      • Habermacher C.
      • Martz A.
      • Calimet N.
      • Lemoine D.
      • Peverini L.
      • Specht A.
      • Cecchini M.
      • Grutter T.
      Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel.
      ,
      • Heymann G.
      • Dai J.
      • Li M.
      • Silberberg S.D.
      • Zhou H.X.
      • Swartz K.J.
      Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels.
      ) also suggested that the TM region was distorted due to the unexpected absence of intersubunit interactions in the X-ray open structure of zfP2X4 receptor. Thus, although the apo/closed and ATP-bound open X-ray structures of the zfP2X4 receptor have provided a blueprint for the mechanism of P2X activation, the detailed conformational transition during channel gating requires further exploration (
      • Kasuya G.
      • Fujiwara Y.
      • Takemoto M.
      • Dohmae N.
      • Nakada-Nakura Y.
      • Ishitani R.
      • Hattori M.
      • Nureki O.
      Structural insights into divalent cation modulations of ATP-gated P2X receptor channels.
      ,
      • Habermacher C.
      • Martz A.
      • Calimet N.
      • Lemoine D.
      • Peverini L.
      • Specht A.
      • Cecchini M.
      • Grutter T.
      Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel.
      ,
      • Wang J.
      • Yu Y.
      Insights into the channel gating of P2X receptors from structures, dynamics and small molecules.
      ,
      • Heymann G.
      • Dai J.
      • Li M.
      • Silberberg S.D.
      • Zhou H.X.
      • Swartz K.J.
      Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels.
      ,
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ,
      • Zhao W.S.
      • Sun M.Y.
      • Sun L.F.
      • Liu Y.
      • Yang Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Cao P.
      • Hu Y.M.
      • Li L.
      • Tian Y.
      • Wang R.
      • Yu Y.
      A highly conserved salt bridge stabilizes the kinked conformation of β2,3-sheet essential for channel function of P2X4 receptors.
      ,
      • Pippel A.
      • Stolz M.
      • Woltersdorf R.
      • Kless A.
      • Schmalzing G.
      • Markwardt F.
      Localization of the gate and selectivity filter of the full-length P2X7 receptor.
      ,
      • Minato Y.
      • Suzuki S.
      • Hara T.
      • Kofuku Y.
      • Kasuya G.
      • Fujiwara Y.
      • Igarashi S.
      • Suzuki E.
      • Nureki O.
      • Hattori M.
      • Ueda T.
      • Shimada I.
      Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region.
      ).
      Figure thumbnail gr1
      Figure 1Bound ATP-evoked allosteric changes associated with channel opening of P2X4 receptors. A, allosteric changes essential for the channel activation of P2X4 receptors. The white dotted lines denote the outward flexing of two lower body domains and the concomitant expansion of the central vestibule of P2X4 receptors. The gray and red arrows indicate the conformational changes after ATP binding and the cation-permeating pathway, respectively. B, superposition of resting (blue) and open (red) conformations of P2X4 receptor and zoom-in view of the expansion of central vestibule. The gray arrows indicate the movements of the DF, LF, and lower body domains associated with the expansion of central vestibule.
      The proposed gating mechanism based on the open and closed X-ray structures highlights that the repelling action of ATP on the LF domain would favor the outward flexing of lower body domains and concomitant pore dilation (
      • Hattori M.
      • Gouaux E.
      Molecular mechanism of ATP binding and ion channel activation in P2X receptors.
      ). The reason why the motion of the LF domain favors this outward flexing of the lower body domains is yet unknown. Recently, it has been demonstrated that ATP binding-induced alteration in interdomain hydrophobic interactions and the concomitant relative motions between the LF and DF domains are indispensable allosteric events for channel activation of P2X4 receptors (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ), although the underlying mechanism of this process remains undetermined. One possibility is that the hydrophobic interactions between the LF and DF domains at the resting state build up an energy barrier that prevents the activation of P2X receptors (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ). The expelling of the LF domain from the ATP-binding pocket might help to overcome this energy barrier and favor the channel activation of P2X4. If so, bound ATP would finally soothe this loop structure to reduce the energy barrier during the channel opening. However, more inter- and intrasubunit contacts among the LF, DF, and lower domains were established after bound ATP pushed the LF domain out of the ATP-binding pocket (details see below and Ref.
      • Hattori M.
      • Gouaux E.
      Molecular mechanism of ATP binding and ion channel activation in P2X receptors.
      ), implying a less flexible LF domain at the open state was developed after the repulsion. Thus, previous studies and structural models (
      • Hattori M.
      • Gouaux E.
      Molecular mechanism of ATP binding and ion channel activation in P2X receptors.
      ,
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ) only partially elucidate the function of the LF domain during channel gating. What exactly is the reason behind the bound ATP-induced repulsion of this flexible loop out of the ATP-binding pocket? Is it a passive allosteric change for only adapting the LF domain to accommodate the allostery of other domains or a more important allostery making a major contribution to couple the ATP binding to the final pore opening? Getting a clear understating of this allostery will provide new mechanical insights into the gating process of P2X4 receptors.
      Using multidisciplinary approaches, we proposed that ATP-bound induced conformational changes of the LF domain enable it to establish intersubunit physical couplings among the DF and two lower body domains, which are essential for the channel opening of P2X4 receptors. This observation will enrich our understandings of the role of the LF domain in channel gating and provide new mechanistic insights into the channel activation of P2X receptors.

      Results

      Intersubunit physical contacts among the lower body and DF domains established by the LF domain are essential for the channel function of P2X4 receptors

      Revealed by the homology models of rat P2X4 (rP2X4) receptors (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ,
      • Zhao W.S.
      • Sun M.Y.
      • Sun L.F.
      • Liu Y.
      • Yang Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Cao P.
      • Hu Y.M.
      • Li L.
      • Tian Y.
      • Wang R.
      • Yu Y.
      A highly conserved salt bridge stabilizes the kinked conformation of β2,3-sheet essential for channel function of P2X4 receptors.
      ) built from zfP2X4 X-ray structures of the apo and open states (
      • Hattori M.
      • Gouaux E.
      Molecular mechanism of ATP binding and ion channel activation in P2X receptors.
      ), the LF domain is a loop structure surrounded by the head, DF, right flipper, and lower body domains (Fig. 2A). Its N and C termini are covalently coupled with the β12 and β13 sheets of the lower body domain (Fig. 2, B and C), respectively. Alanine-scanning mutagenesis of all residues of the LF domain (Fig. 2, D and E), ranging from Arg-277 to Tyr-292 of rP2X4, indicated that the residues in the N and C termini rather than those in the middle region, including Arg-278, Leu-279, Asp-280, Arg-282, and Pro-290 (Fig. 2, D and E), are essential for the channel function of P2X4 receptors. Mutations on identical residues in zfP2X4 (Arg-281, Asp-283, and Lys-285, Fig. 3A) significantly reduced ATP (1 mm)-induced currents (Fig. 3, B and C), indicating a crucial role of these residues in both rP2X4 and zfP2X4.
      Figure thumbnail gr2
      Figure 2Intersubunit physical contacts fostered by the LF domain at the open state are essential for rP2X4 activation. A, 3-D homology model of rP2X4. LF domain locates in the interface between two subunits and is surrounded by the head, DF, and two lower body domains. B and C, zoom-in views of the 3-D structure of N (B) and C termini (C) at resting (upper) and open (lower) states of P2X4 receptor illustrate the switching of H-bonds (red dotted lines) between key residues in the LF, DF, and lower body domains before and after ATP binding. D, representative raw traces of P2X4's responses to saturated ATP (1–1.5 mm for R278A, D280A, R282A, P290A, and R203A; 100 μm for WT and other mutants). E, pooled data of ATP-evoked maximal current amplitudes in P2X4 with alanine replacements on the residues of LF domain (mean ± S.E., n = 4–20). ∗, p ≪ 0.05; ∗∗, p ≪ 0.01 versus WT (dashed line), one-way ANOVA with Bonferroni post hoc test.
      Figure thumbnail gr3
      Figure 3Intersubunit physical contacts fostered by the LF domain at the open state are essential for zfP2X4 activation. A, zoom-in view of the 3-D structure of the LF domain at the open state of zfP2X4 receptors (PDB code 4DW1) illustrates the H-bonds (red dotted lines) between key residues in the LF, DF, and lower body domains after ATP binding. B and C, representative raw traces (B) and pooled data (C) of WT zfP2X4 and various mutant (R281A, D283A, K285D, and R206L) responses to ATP (1 mm, mean ± S.E., n = 3–7). ∗, p ≪ 0.05; ∗∗, p ≪ 0.01 versus WT (dashed line), one-way ANOVA with Bonferroni post hoc test. D, amino acid sequence alignment of the LF and lower body domains in different P2X subtypes. In the amino acid sequence alignment, the residues were numbered according to the amino acid sequence of rP2X4 receptors, and N, C, and M refer to the N and C termini and middle region of the LF domain, respectively.
      A comparison of the LF domain at the open and closed states based on the homology models of rP2X4 revealed that all mutants impairing the maximal current amplitude virtually contribute to the newly established intersubunit physical couplings among the two lower body and DF domains after the LF domain was repelled out of the ATP-binding site (Fig. 2, B and C). Arg-278 and Asp-280 foster an intrasubunit salt bridge in both the closed and open structures of P2X4, whereas an additional hydrogen bond (H-bond) was formed between the side chain of Arg-282 and the oxygen atom of the main chain of Arg-278 at the open state (Fig. 2B, lower panel). ATP binding also contributes to the formation of the H-bond between the side chain of Asn-192 (located in the lower body domain of another subunit) and the main chain atom of Arg-282 (Fig. 2B, lower panel). N192A mutant partially reduced the maximal current amplitude of P2X4 (Fig. 2, D and E). Additionally, the atoms of the main chain of Val-288, Ser-289, and Pro-290 developed new contacts with the side chain of Arg-203 (in the lower body domain of another subunit) (Fig. 2C, lower panel). Alanine substitution of Arg-203 significantly impaired channel activation of rP2X4 receptors (Fig. 2, D and E). Mutations on the identical residue (Arg-206) in zfP2X4 significantly reduced ATP (1 mm)-induced currents (Fig. 3, B and C), suggesting this newly established contact is also crucial for the channel function of zfP2X4 receptors. The intersubunit hydrophobic contacts among Val-288, Leu-214, and Ile-205 were significantly changed after ATP binding (Fig. 2C) (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ). Mutations on Val-288, Leu-214, Ile-205, and the identical residues of zfP2X4 significantly reduced maximal current densities of both rP2X4 and zfP2X4 receptors (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ). In contrast, although Glu-245 forms intersubunit contact with Arg-282 via H-bonds to tighten the LF domain at the resting state (Fig. 2B, upper panel), E245A and E245R had no effect on the maximal current amplitude and the EC50 of ATP (the concentration of ATP yielding current that is half of the maximum) of rP2X4 (see below), indicating that this contact is redundant for the channel function. Thus, ATP binding-induced conformational changes of the LF domain and the following intersubunit physical couplings among the DF and two lower body domains fostered by the deformed LF domain at the open state are pivotal for the channel function of both rP2X4 and zfP2X4 receptors.
      Because of the low potency of ATP on the zfP2X4 receptor expressing in mammalian cells (
      • Kawate T.
      • Michel J.C.
      • Birdsong W.T.
      • Gouaux E.
      Crystal structure of the ATP-gated P2X(4) ion channel in the closed state.
      ,
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ,
      • Zhao W.S.
      • Sun M.Y.
      • Sun L.F.
      • Liu Y.
      • Yang Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Cao P.
      • Hu Y.M.
      • Li L.
      • Tian Y.
      • Wang R.
      • Yu Y.
      A highly conserved salt bridge stabilizes the kinked conformation of β2,3-sheet essential for channel function of P2X4 receptors.
      ), the following mutagenesis, protein-expression measurements, and electrophysiological recordings were carried out on rP2X4 receptors. Additionally, although the essential role of the residues involved in forming intersubunit physical couplings after ATP binding was conserved between rP2X4 and zfP2X4 receptors (Fig. 3, A and D) (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ), some residues of the middle region of the LF domain are not exactly the same (Fig. 3D). Thus, to get a more appropriate prediction about the functional studies of the rP2X4 receptors, the following free-energy profile reconstructions and MD simulations were also based on the homology models of rP2X4 rather than on the crystal structures of the zfP2X4 receptor.

      Impaired intersubunit physical couplings significantly influence channel gating rather than channel assembly, protein stability, and ATP-EC50 of P2X4 receptors

      Multiple factors can influence the maximal current densities of P2X receptors. The fact that the surface expression levels of these loss-of-function mutants exhibited no pronounced changes when compared with wild-type (WT) rP2X4 (Fig. 4, A and B) suggests that the decreased ATP currents were not acting on channel expression and trafficking, except for R278A and D280A. The complete abolishment of the ATP-induced currents in rP2X4R278A and rP2X4D280A (Fig. 2, D and E) may partially attribute to a decreased surface expression of those two mutants. Another possibility is that the LF domain and the established intersubunit physical couplings may structurally support the trimeric assembly or prevent the neighboring subunit from clashing with each other upon ATP binding. We introduced additional mutations, R203A and R282A, into mutant V288C/T211C, which could form trimeric receptors in non-reduced SDS-PAGE, due to the formation of intersubunit disulfide bonds as we previously demonstrated (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ). Trimeric bands of mutants, V288C/T211C/R203A and V288C/T211C/R282A, were observed in the non-reducing Western blotting (Fig. 4C), indicating that the impaired intersubunit physical couplings did not render a significant deficiency in the channel assembly of P2X4 receptors. Additionally, we incubated the transfected HEK-293 cells with cycloheximide (CHX, 20 μg/ml), an inhibitor of protein biosynthesis (
      • Satav J.G.
      • Katyare S.S.
      • Fatterparker P.
      • Sreenivasan A.
      Study of protein synthesis in rat liver mitochondria use of cycloheximide.
      ), in time-scale experiments. In contrast to the profound reduction in maximum currents, after incubation with CHX for 10 h, the protein level of the mutants made no significant changes (Fig. 4, D and E), indicating that alterations in those positions did not render channel instability of P2X4 receptors. Moreover, in contrast to the profound reduction in maximum currents (Fig. 4, F and G), little to no change was observed in the surface expression (Fig. 4, H and I) and EC50 values for the functional mutants, namely R203K (EC50 = 1.43 ± 1.1 μm) and R282W (1.44 ± 1.2 μm), compared with WT channels (EC50 = 1.96 ± 1.2 μm) (Fig. 4J). Thus, the decreased maximal current densities in those mutants were not related to surface protein expression, channel assembly, protein stability, or EC50 of ATP.
      Figure thumbnail gr4
      Figure 4Effects of mutants on the channel functions of rP2X4 receptors. A and B, representative Western blotting (A) and mean values (B) of the membrane expression of P2X4 with alanine replacements transfected in HEK-293 cells. At least three experiments were performed for each mutant: ∗, p ≪ 0.05; ∗∗, p ≪ 0.01 versus WT, one-way ANOVA with Bonferroni post hoc test. C, protein samples extracted from transfected HEK-293 cells separated by non-reducing Western blotting. Monomeric, dimeric, and trimeric receptors are indicated by triangle arrows on the left. Molecular mass markers are shown on the right. Similar results were observed in at least three independent experiments. D and E, representative Western blotting (D) and mean values (E) of rP2X4 protein for WT, R203A, R278A, D280A, and R282A. Cells were treated with 20 μg/ml CHX in a time-course experiment as indicated. The results were observed in at least three independent experiments for statistical analysis. F and G, representative traces (F) and mean values (G) of the responses of WT rP2X4 and various mutants to ATP (100 μm, mean ± S.E., n = 3–7). ∗, p ≪ 0.05; ∗∗, p ≪ 0.01 versus WT (dashed line), one-way ANOVA with Bonferroni post hoc test. H and I, representative Western blotting (H) and mean values (I) of the membrane protein expression for WT, R282W, and R203K. At least three experiments were performed for each mutant: ∗, p ≪ 0.05; ∗∗p ≪ 0.01 versus WT, one-way ANOVA with Bonferroni post hoc test. J, effects of mutations on the ATP-EC50 of rP2X4. The solid line is a fit of the Hill equation to the ATP-dependent activation. Each point represents the mean ± S.E. of four measurements.
      To gain more structural information about the role of those newly established intersubunit physical couplings in the channel function of P2X4 receptors, the free-energy profiles for those interactions were reconstructed by metadynamics (
      • Laio A.
      • Parrinello M.
      Escaping free-energy minima.
      ,
      • Laio A.
      • Rodriguez-Fortea A.
      • Gervasio F.L.
      • Ceccarelli M.
      • Parrinello M.
      Assessing the accuracy of metadynamics.
      • Limongelli V.
      • Bonomi M.
      • Marinelli L.
      • Gervasio F.L.
      • Cavalli A.
      • Novellino E.
      • Parrinello M.
      Molecular basis of cyclooxygenase enzymes (COXs) selective inhibition.
      ), “a powerful algorithm that can be used for both reconstructing the free energy and accelerating rare events in systems described by complex Hamiltonians” (
      • Laio A.
      • Gervasio F.L.
      Metadynamics: a method to simulate rare events and reconstruct the free energy in biophysics, chemistry and material science.
      ). The N-O distances of Arg-282…Glu-245 and Arg-203…Val-288 are distinct between the resting and open states (Fig. 5A). After ATP binding, Arg-282 moved away from the Glu-245 (N-O distances of Arg-282–Glu-245 = 4.6 and 14.9 Å, at the resting and open states, respectively), whereas Arg-203 moved closer to Val-288 (N-O distances = 11.4 and 3.1 Å, at the resting and open states, respectively), indicating new intersubunit contacts among these residues were established. These two distance measurements were defined as collective variables (CVs) of metadynamics (Fig. 5A). The lower free-energy paths (gray line) plotted onto free energy profiles passing those two CVs from open to resting states were carried out under the conditions with (Fig. 5B) and without (Fig. 5C) bound ATP to figure out the correlation between these allosteric changes and the ATP-bound open state. The bound ATP made this passing difficult due to the high energy barrier existing in the passing path (Fig. 5B), indicating that the existence of both the bound-ATP and new intersubunit contacts had “locked” the P2X4 receptor at the open state. In contrast, when the bound ATP was taken away from the ATP-binding site (Fig. 5C), this passing became spontaneous because free-energy profiles revealed a more stable CV similar to that of the resting state (CVR) than that of open state (CVO), indicating that the LF domain-mediated establishment of intersubunit physical couplings is an allosteric change correlated well with the ATP-bound open state. Because these mutations had little to no changes in the EC50 of ATP, the protein expression and channel assembly, despite profound reduction in maximum currents (Fig. 4), they may impair the channel gating of P2X4 receptors.
      Figure thumbnail gr5
      Figure 5Impaired intersubunit physical couplings significantly influence channel gating of P2X4 receptors. A, N-O distances of Arg-282…Glu-245 (CV1) and Arg-203…Val-288 (CV2) at the resting (left) and open (right) states. Two measured distances were defined as collective variables (CVs) of metadynamics. B and C, 3-D projection of free-energy (kcal mol−1) surface showing the lowest free-energy paths passing from open to resting state with (B) or without (C) bound-ATP. CVO and CVR indicates CVs at the open and resting states, respectively. The gray dashed lines and white arrowheads depict the paths passing from resting to open state. D–F, single channel currents recorded from outside-out patches at −120 mV in responses to ATP (100 μm) for the WT (D), R203A (E), and R282A (F). Full opening (O) and closing (C) are indicated by black and yellow lines, respectively. Right panel summarizes corresponding all-points histograms fitted to the sum of two Gaussians. y axis denotes the ratio of the number of events to the number of bins (the bin number is set to 320). Similar results were obtained in four other independent patches. G, single channel currents recorded from outside-out patches at −120 mV in responses to ATP and following ATP-IVM co-application for the mutants R203A, R282A, and WT rP2X4. Similar results were obtained in at least three other independent recordings. H, mean amplitude of the unitary current of WT rP2X4 and mutants in the presence of ATP and IVM.
      To test this hypothesis, we performed single channel recordings in the outside-out configuration. The control experiment showed that the main conductance state of channels opened by saturated ATP (100 μm) in patches excised from HEK-293 cells expressing WT rP2X4 (Fig. 5D) was similar to that shown in previous reports (
      • Evans R.J.
      Single channel properties of ATP-gated cation channels (P2X receptors) heterologously expressed in Chinese hamster ovary cells.
      ,
      • Negulyaev Y.A.
      • Markwardt F.
      Block by extracellular Mg2+ of single human purinergic P2X4 receptor channels expressed in human embryonic kidney cells.
      • Priel A.
      • Silberberg S.D.
      Mechanism of ivermectin facilitation of human P2X4 receptor channels.
      ). The channel with a unitary conductance of ∼10 pS at −120 mV was observed in the most of the patches. Channels opened and closed frequently and were highly flickery, and thus the precise determination of the open and shut time could not be made. Additionally, unitary currents could only be observed at the first 5 s and disappeared after ∼10 s (Fig. 5D). By contrast, there were no or only few channel openings resembling unitary rP2X4 current in rP2X4R203A (Fig. 5E) and rP2X4R282A (Fig. 5F) in response to saturated ATP (100 μm). When ATP (100 μm) was co-applied with ivermectin (IVM) (3 μm), a widely used P2X4 enhancer (
      • Priel A.
      • Silberberg S.D.
      Mechanism of ivermectin facilitation of human P2X4 receptor channels.
      ,
      • Khakh B.S.
      • Proctor W.R.
      • Dunwiddie T.V.
      • Labarca C.
      • Lester H.A.
      Allosteric control of gating and kinetics at P2X(4) receptor channels.
      • Silberberg S.D.
      • Li M.
      • Swartz K.J.
      Ivermectin interaction with transmembrane helices reveals widespread rearrangements during opening of P2X receptor channels.
      ), unitary currents could be observed both in rP2X4R203A and rP2X4R282A (Fig. 5G). Similar to a previous finding (
      • Priel A.
      • Silberberg S.D.
      Mechanism of ivermectin facilitation of human P2X4 receptor channels.
      ), IVM exhibited a small effect on the unitary current amplitude (Fig. 5, G and H) of rP2X4R203A, rP2X4R282A, and WT and significantly prolonged the open time of channels (Fig. 5G). Perhaps this is because IVM directly acts on the interface between transmembrane domains TM1 and TM2 (
      • Silberberg S.D.
      • Li M.
      • Swartz K.J.
      Ivermectin interaction with transmembrane helices reveals widespread rearrangements during opening of P2X receptor channels.
      ), the only entrance of various ions. The generation of unitary currents of rP2X4R203A and rP2X4R282A in the patches exposed to IVM and ATP further supported the idea that the mutated channels with impaired intersubunit physical couplings fostered by the LF domain behave with normal surface expression, channel assembly, and the EC50 of ATP. It has been well established that IVM is able to significantly increase the channel opening probability (Po) of P2X4 receptors (
      • Priel A.
      • Silberberg S.D.
      Mechanism of ivermectin facilitation of human P2X4 receptor channels.
      ). Thus, the absence of unitary P2X4 currents in mutated channels exposed to saturated ATP and the significant channel activity observed in the presence of both ATP and IVM suggested that the impairment in intersubunit physical couplings significantly decreased the open probability of P2X4 receptors.

      Engineered intersubunit metal bridges that change the intersubunit physical couplings remarkably influence the channel gating of P2X4 receptors

      To further examine this idea, we applied different metal bridges to weaken or strengthen the physical couplings among the DF and two lower body domains fostered by the LF domain at the open state. We have recently shown that an intradomain Zn2+ bridge in the LF domain produces an unexpected inhibition (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ) on the current of mutant channel rP2X4His-286/V288H without identifying the underlying mechanism. Here, the structural model of rP2X4His-286/V288H in the open state suggested that Zn2+ might form an intersubunit rather than an intrasubunit Zn2+ bridge with the introduced histidine residue V288H and natural histidine residue His-286 of one subunit, as well as with the main chain oxygen atoms of Pro-207 and Ile-209 of another subunit (Fig. 6A). Indeed, post-application of Zn2+ after ATP markedly reduced the remaining currents of rP2X4His-286/V288H (ratio = 0.26 ± 0.06, n = 4, green arrow, Fig. 6, B and C). This blockage was specific to the presence of histidine at both 288 and 286 positions because Zn2+ application only caused mild inhibition on the single histidine mutant H286A/V288H as well as WT P2X4 (His-286/Val-288) receptor (Fig. 6C). Introduction of the additional mutation into His-286/V288H (His-286/V288H/P207A and His-286/V288H/I209A) affected both the inhibition (Fig. 6C) and the dose-response curve of Zn2+ (Fig. 6D) of rP2X4 (IC50, the half-inhibition of Zn2+ = 68.4 ± 10, 39.4 ± 2, and 129.8 ± 17 μm, for rP2X4His-286/V288H, rP2X4His-286/V288H/P207A, and rP2X4His-286/V288H/I209A, respectively), indicating that Pro-207 and Ile-209 may contribute to the intersubunit binding of Zn2+ at the open state.
      Figure thumbnail gr6
      Figure 6Introducing different intersubunit metal bridges to weaken or strengthen intersubunit physical couplings at the open state. A, zoom-in view of the constructed zinc-bridge model of P2X4His-286/V288H based on the open structure provides the details of the distances (blue dotted line) between Zn2+ and the coordinating NE2 or oxygen atoms from His-286, His-288, Pro-207, and Ile-209. The black circles denote the Cα atoms of residues His-288, Arg-278, and Ile-205 of P2X4His-286/V288H. The Cα atoms of residues Arg-278 and Ile-205 align horizontally, whereas the Cα atoms of His-288 and Arg-278 form an angle to Arg-278 and Ile-205, suggesting a tilting posture of the LF domain. The degree of the angle correlates with the intersubunit physical couplings between DF and two lower body domains established by deformed LF domain at the open state. Red arrow indicates the downward motion of LF domain of P2X4His-286/V288H after Zn2+ application. B and C, sample traces (B) and summarized (C, mean ± S.E., n = 4–12) effects of extracellular Zn2+ treatment on ATP (100 μm, saturating)-evoked currents of WT and mutant receptors. ∗∗, p ≪ 0.01 versus P2X4 WT; #, p ≪ 0.05 versus control, one-way ANOVA followed by Bonferroni post hoc test. D, dose-response curves of Zn2+ in P2X4His-286/V288H, P2X4His-286/V288H/P207A, and P2X4His-286/V288H/I209A fitted into Hill (solid line, IC50 = 68.4 ± 10, 39.4 ± 2, and 129.8 ± 17 μm, for P2X4His-286/V288H, P2X4His-286/V288H/P207A, and P2X4His-286/V288H/I209A, respectively). Data points are mean ± S.E. of 5–10 measurements. E, time evolutions of the tilting angle of LF domain along the horizontal line (measured by angle formed by Cα atoms of residues 288, 278, and 205) during MD simulations on WT, P2X4His-286/V288H, and P2X4His-286/V288H/I209C. Zn2+ binding significantly decreased the tilt angle of the LF domain in P2X4His-286/V288H but increased in mutant His-286/V288H/I209C when compared with that of WT P2X4 receptor. F, zoom-in view of the constructed zinc-bridge model of P2X4His-286/V288H/I209C based on the open structure showing details of the distances (light-blue dotted line) between Zn2+ and the coordinating NE2, sulfur, and oxygen atoms of His-286, His-288, and Cys-209.
      The structural model of rP2X4V288H/His-286 at the open state revealed that the Zn2+ bridge between His-286, V288H, and the oxygen atoms of the main chain of Pro-207 and Ile-209 is capable of trapping the C-terminal of the LF domain into the flexible loop β9-α3 (Fig. 6A, right panel). This idea is further supported by the measurement of the angle between three Cα atoms of residues His-288, Arg-278, and Ile-205 of P2X4V288H/His-286 (Fig. 6, A and E). The Cα atoms of residues Arg-278 and Ile-205 align horizontally, whereas the Cα atoms of His-288 and Arg-278 form an angle to Arg-278 and Ile-205, suggesting a tilted shape of the LF domain (Fig. 6, A and E). The tilting angle of the LF domain along the horizontal axis in P2X4His-286/V288H ranged from 23–25° (Fig. 6E) during MD simulations, a value smaller than that of WT rP2X4 (25–28°) (Fig. 6E), indicating that the C terminus moved downwards and became closer to the flexible loop β9-α3 than WT rP2X4 (Fig. 6A). Because the N terminus of the LF domain was located in the rigid lower body domain (β12) of another subunit (Fig. 6A), the C-terminal movement toward the flexible loop β9-α3 significantly weakened intersubunit physical couplings between the DF and two lower body domains fostered by the deformed LF domain after ATP binding. To prevent this movement, we introduced an additional substitution in loop β9-α3 (His-286/V288H/I209C) to push the C terminus away from the flexible loop β9-α3 through an additional coordination bond between Zn2+ and the free thiol group of I209C (Fig. 6F), and to regain contacts between the C terminus and rigid the α3 helix of the DF domain (Fig. 6F). This additional replacement on Ile-209 by cysteine significantly increased the angle formed by Cα atoms of residues His-288, Arg-278, and Ile-205 of rP2X4His-286/V288H/I209C during MD simulations (Fig. 6, E and F), indicating that the LF domain had escaped from the flexible loop β9-α3 and produced more contacts with the rigid α3 helix of the DF domain (Fig. 6F, right lower panel).
      Indeed, the additional application of Zn2+ after ATP led to a remarkable potentiation (ratio = 3.55 ± 0.36, n = 12) rather than an inhibition on the remaining current of His-286/V288H/I209C (right trace of Fig. 6, B, green arrow, and C, green column), revealing the pivotal role of intersubunit physical couplings between the DF and two lower body domains established by the LF domain in the open state. This potentiation requires the presence of both histidine residues His-288 and His-286 in the LF domain of one subunit, and the Cys-209 located in the interface between the DF and lower body domains of another subunit, because Zn2+ application only slightly inhibited rather than potentiated the currents of H286A/V288H/I209C, His-286/Val-288/I209C, and H286A/Val-288/I209C (Fig. 6C).
      The effect of Zn2+ on the unitary rP2X4His-286/V288H/I209C currents was also measured in the outside-out configuration of patch clamp. There were no channel openings resembling unitary rP2X4 currents in the presence of saturated ATP (Fig. 7A), although currents with a unitary conductance of ∼10 pS at −120 mV were evoked when ATP and Zn2+ were co-applied (Fig. 7A). In additional macroscopic recordings, a few unitary P2X4 currents (∼10 pS) were observed when only 100 μm ATP was applied (Fig. 7B); however, a following co-application of ATP and Zn2+ evoked a large current (∼30 channels simultaneously opening) that rapidly declined to a steady-state level, where individual openings and closings can be measured, indicating that the engineered metal bridge rendered a significant increase in the open probability of rP2X4 receptors but had no remarkable effects on the rP2X4 desensitization. The unitary current conductance was similar before (∼10 pS) and after Zn2+ treatment (∼10 pS, Fig. 7, A and the lower panel of B), suggesting these physical couplings had no effect on the unitary current conductance of rP2X4 receptors. Thus, physical couplings among the DF and two lower body domains fostered by the LF domain increase the open probability rather than change the current unitary conductance and channel desensitization of rP2X4 receptors.
      Figure thumbnail gr7
      Figure 7Effect of Zn2+ on the unitary rP2X4His-286/V288H/I209C currents. A and B, representative current recordings from excised outside-out membrane expressing single channel (A) or multiple channels (B) exposed to ATP and the following ATP-Zn2+ co-application for the mutant His-286/V288H/I209C. Full opening (O) and closing (C) are indicated by black and yellow lines, respectively. y axis denotes the ratio of the number of events to the number of bins (the bin number is set to 320). Similar results were obtained in at least three other independent recordings.

      Restraining the LF domain from fostering physical couplings via intersubunit disulfide cross-linking impairs channel activation of P2X4 receptors

      To further examine the contribution of the intersubunit physical couplings fostered by the deformed LF domain during the channel gating of rP2X4 receptors, we immobilized the LF domain at the resting state by introducing cysteine residues that form the intersubunit disulfide bridge (Fig. 8A). The Cβ–Cβ distances of Ser-201–Asp-283 (14.2 Å versus 7.1 Å, resting versus open states) and Ser-201–Leu-284 (19.4 Å versus 7.9 Å, resting versus open states) (Fig. 8, B and C) were longer than that of a disulfide pair (≪5 Å) (
      • Cremlyn R.J.
      ). Therefore, the interdomain disulfide bond at those positions could immobilize the LF domain at the resting state and restrain the LF domain from fostering intersubunit physical coupling at the open state (Fig. 8A). The WT rP2X4 and single mutants S201C, D283C, and L284C migrated on SDS-polyacrylamide gels predominantly at a position expected for monomeric form (∼57 kDa; Fig. 8D). In contrast, no obvious monomeric form was observed for the subunits containing cysteine at both positions (S201C/D283C and S201C/L284C). The observed higher molecular weights presumably represent disulfide bond trimer, because β-mercaptoethanol (β-ME, 1%) reduced those to a monomeric size (Fig. 8D). Although β-ME caused a modest shift of WT and single cysteine replacement mutants as we previously reported because of its effects on the large number of native cysteine residues (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ), it should still be reasonable to conclude that interdomain/intersubunit disulfide bonds are actually formed between the LF and lower body domains in S201C/D283C and S201C/L284C.
      Figure thumbnail gr8
      Figure 8Restraining the LF domain from fostering physical couplings via intersubunit disulfide. A, intersubunit disulfide bonds between the LF and lower body domains immobilize LF domain at the resting state and prevent the establishment of intersubunit physical couplings at the open state. B and C, zoom-in view of the Cβ−Cβ distances (green dotted line) between Ser-201 and the key residues of the middle region of the LF domain at the resting (B) and open (C) states. D, Western blotting results support the formation of intersubunit disulfide bonds between S201C (in the lower body domain of one subunit) and D283C/L284C (in the LF domain of another subunit) in homooligomeric P2X4 receptors. The cells transfected with WT or cysteine substitution mutants were lysed in buffer with or without β-ME (1%, 10 mm) as indicated. Positions corresponding to the size of monomeric, dimeric, and trimeric P2X4 subunits were labeled with arrowheads, respectively. E and F, representative currents recorded from cells transfected with S201C/D283C and S201C/L284C (E) and WT P2X4 receptors (F). Cells were voltage-clamped at −60 mV and currents were evoked by ATP (10 μm, 3 s) at 2-min intervals. DTT (10 mm) and H2O2 (0.3%) were applied as the schematic indicated. G, pooled data from the experiments in E and F. y axis denotes the ratio of ATP-evoked current after DTT treatment normalized by current before DTT application (mean ± S.E., n = 3–12). ∗∗, p ≪ 0.01 after DTT application versus before DTT application, paired t test. H and I, representative recordings to effects of DTT (10 mm) and H2O2 (0.3%) on lower concentrations (H) and saturated (I) ATP-sensing of P2X4S201C/D283C. J, normalized P2X4S201C/D283Cand P2X4S201C/L284C currents evoked by saturated ATP. y axis denotes the ratio of ATP-evoked current after DTT treatments normalized by current before DTT applications (mean ± S.E., n = 3). ∗∗, p ≪ 0.01 after versus before, paired t test.
      As a result, after expression in HEK-293 cells, the mutated channels with double cysteine substitutions (S201C/D283C, 4.06 ± 1.7 pA/pF, n = 7; and S201C/L284C, 6.39 ± 1.3 pA/pF, n = 8) produced much smaller responses to ATP when compared with WT (249 ± 25 pA/pF, n = 20) and mutants with single cysteine replacements S201C (91.7 ± 37 pA/pF, n = 6), D283C (145 ± 37 pA/pF, n = 7), and L284C (137 ± 53 pA/pF, n = 7). Applications of dithiothreitol (DTT) increased the ATP-induced currents of S201C/D283C and S201C/L284C for ∼4–5- and ∼2–3-fold (ratio = 4.49 ± 0.67, n = 3, p = 0.003 and 2.19 ± 0.39, n = 4, p = 0.006), respectively (Fig. 8, E, G, and H), which were reversed by applications of H2O2 (Fig. 8E), suggesting that immobilization of the LF domain led to impaired channel activation of P2X4. In contrast, DTT slightly reduced ATP-evoked currents of the WT rP2X4 receptors, which was reversed by H2O2 (Fig. 8F). All the results suggested that the breaking of disulfide bonds is responsible for DTT-induced increases in current amplitudes of S201C/D283C and S201C/L284C. Additionally, increasing ATP concentration (1 mm) had no effect on DTT-induced potentiation efficacy on rP2X4S201C/D283C and rP2X4S201C/L284C currents (Fig. 8, G–J) (S201C/D283C: 4.5 ± 0.7- and 4.4 ± 0.8-fold for 10 μm and 1 mm ATP-induced currents, n = 3 and 3, respectively, p = 0.93, t test; S201C/L284C: 2.2 ± 0.4- and 1.9 ± 0.3-fold potentiation for 10 μm and 1 mm ATP-induced currents, n = 4 and 3, respectively, p = 0.59, t test). Thus, the impaired channel gating of rP2X4S201C/D283C and rP2X4S201C/L284C by disulfide cross-linking is not related to alterations in ATP sensing.
      However, disulfide cross-linking may interrupt both conformations of the LF domain at resting and open states. To provide direct evidence that restraining the LF domain from fostering intersubunit physical couplings at the open state can influence channel activation of P2X4 receptors, we introduced an intersubunit Zn2+ bridge between the LF and lower body domains to slightly perturb the conformation of the middle region of the LF domain at the open state (Fig. 9A). This Zn2+ bridge at the open state was established via DTT treatments (10 mm for 10 min) at the resting state of rP2X4S201C/D283C, which enables the free cysteine residues Cys-201 and Cys-283 to chelate Zn2+ at the open state. The Cβ–Cβ distance of the two free cysteine residues in the natural Zn2+ bridge of crystal structures ranges from 4.5 to 5.0 Å (
      • Alberts I.L.
      • Nadassy K.
      • Wodak S.J.
      Analysis of zinc binding sites in protein crystal structures.
      ), a value relatively smaller than Cβ–Cβ distance of Ser-201 and Asp-283 (7.1 Å, Fig. 8C) at the open state. Therefore, a rebuilt Zn2+ bridge (Cys-201…Zn2+…Cys-283, Fig. 9A; Cβ–Cβ distance of Cys-201 and Cys-283 = 5.2 Å) after DTT application on P2X4S201C/D283C could perturb the conformation of the middle region of the LF domain. Indeed, post-administration of Zn2+ after ATP inhibited 51.7 ± 2.3% of the remaining ATP currents of P2X4S201C/D283C but not WT P2X4 (n = 9, p = 0.0006, Fig. 9, B and C) under the condition that the disulfide bond has been previously interrupted by DTT at the resting state. This point was further tested by a measurement of state-dependent cross-linking of rP2X4S201C/D283C. After DTT breaking, the cross-linking between S201C and D283C rebuilt more quickly after rP2X4S201C/D283C was treated by ATP, when it is compared with channels without ATP treatment (Fig. 9D). Alanine-scanning mutations on the LF domain have demonstrated that the residues of the middle region (Figs. 2, D and E, and 10A) were not as crucial as the residues in the N- and C-terminal regions in the channel activation of P2X4 receptors. However, an immobilization of the LF domain using disulfide cross-linking or slightly shortening the pair-residue distance (from 7.1 to 5.2 Å) between the middle region of the LF and lower body domains using a metal bridge rendered a significantly impaired channel activation of rP2X4 receptors. Therefore, intersubunit physical couplings among the DF and two lower body domains fostered by the LF domain at the open state act as a whole structural element that is stringently required by the channel opening of P2X4 receptors, and any impairment in its integrity will lead to an impaired channel activation.
      Figure thumbnail gr9
      Figure 9Formation of intersubunit disulfide perturbs the conformation of the middle region of the LF domain at the open state. A, zoom-in view of the constructed zinc-bridge model of P2X4S201C/D283C based on the open structure exhibits the details of the Cβ–Cβ distances (green dotted line) of Cys-201 and Cys-283 and the distances measured between Zn2+ and the coordinating sulfur atoms from Cys-201 and Cys-283 (yellow dotted line). B and C, sample traces (B) and summarized (C, mean ± S.E., n = 5–9) effects of extracellular Zn2+ treatment on ATP (100 μm, saturated)-evoked remaining currents of WT and S201C/D283C. ∗∗, p ≪ 0.01 after versus before Zn2+ application, paired Student's t test. D, state-dependent cross-linking of rP2X4S201C/D283C. DTT (+/−) indicate with (+) or without (−) the treatment of DTT (10 mm for 10 min) after the cell surface biotinylation; ATP (+/−) indicate with (+) and without (−) a treatment of ATP (100 μm for 1 min) before the cell lysis; β-ME (+/−) indicate the presence (+) and absence (−) of β-ME (1%, 10 mm) in the loading buffer.
      Figure thumbnail gr10
      Figure 10Middle region of the LF domain is a functional structural element in the channel activation of rP2X4 receptors. A, superposition of the closed (purple) and open (pink) conformations of rP2X4. Yellow arrow denotes the motion of the LF domain from closed to ATP-bound open state. The residues of the middle region of the LF domain are displayed as sticks for emphasis. B, representative traces of currents evoked by saturated ATP (1–1.5 mm for Δ283–286; 100 μm for WT and the rest of the mutants). C, pooled data for maximal current amplitude evoked by saturated ATP of mutations on the middle region of the LF domain (mean ± S.E., n = 4–20). ∗, p ≪ 0.05; ∗∗, p ≪ 0.01 versus WT (dashed line), one-way ANOVA with Bonferroni post hoc test. D and E, representative Western blotting (D) and pooled data (E) illustrating the membrane expressions of WT and Δ283–286 in HEK-293 cells. The results were observed in at least three independent experiments.
      Moreover, deletions of a single amino acid (Δ283, Δ284, Δ285, Δ286, and Δ287), two amino acids (Δ283–284, Δ284–285, Δ285–286, and Δ286–287), and even three amino acids (Δ283–285, Δ284–286, and Δ285–287) in the middle region produced little change on the maximal current of P2X4 receptors (Fig. 10, B and C). However, truncating four amino acids (Δ283–286) fully abolished the channel activation of P2X4 (n = 8, Fig. 10, B and C), without changing the channel expression and trafficking of P2X4 receptors (Fig. 10, D and E). Thus, a proper length of the middle region (at least two residues remained among those of five amino acids) is prerequisite for the LF domain being functional, further confirming that the middle region is a structural element required by the P2X4 receptors at the open state (see “Discussion”).

      Weakening intersubunit physical couplings via covalent modifications impairs the channel activation of P2X4 receptors

      Finally, we interrupted the conformation of the N terminus of the LF domain through covalent modifications to partially weaken these intersubunit physical couplings at the open state (Fig. 11A). Upon channel activation, the intrasubunit salt bridge between Arg-278 and Asp-280 as well as the two H-bonds (Arg-282…Arg-278 and Arg-282…Asn-192) formed an “interaction network” that was crucial for the deformation of the LF domain at the open state (Fig. 2B, lower panel). Trp-194 is located adjacent to those four amino acids (Fig. 11A and the lower panel of Fig. 2B). We substituted Trp-194 with cysteine with the intention to prevent the interactions between Arg-278, Asp-280, and Arg-282 via covalent modifications (Fig. 11A). Using a cysteine modification technique such as methanethiosulfonate (
      • Samways D.S.
      • Khakh B.S.
      • Dutertre S.
      • Egan T.M.
      Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors).
      ), Ellman's (
      • Yu Y.
      • Chen Z.
      • Li W.G.
      • Cao H.
      • Feng E.G.
      • Yu F.
      • Liu H.
      • Jiang H.
      • Xu T.L.
      A nonproton ligand sensor in the acid-sensing ion channel.
      ), and alkylating reagents (
      • Aboul-Enein H.Y.
      • Refaie M.O.
      • El-Gazzar H.
      • El-Aziz M.A.
      Chemical modification of milk xanthine oxidase with different modifiers.
      ) (Fig. 11B), we introduced charged groups (the aminoethyl group of 2-aminoethyl methanethiosulfonate (MTSEA) and sulfonatoethyl group of 2-sulfonatoethyl methanethiosulfonate (MTSES)), a bulky group (1-phenylpyrrolidine-2,5-dione group of N-phenylmaleimide (NPM)), and both charged and bulky groups (5-thio-2-nitrobenzoic acid group of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)) into the position (Cys-194) around Arg-278, Asp-280, and Arg-282 in P2X4W194C. Applications of MTSEA (1 mm), MTSES (1 mm), DTNB (1 mm), and NPM (1 mm) markedly reduced the current amplitude of P2X4W194C (Fig. 11, C and D), which were partially or fully reversed by DTT treatments (Fig. 11C), except NPM treatments, because the alkylating reaction is irreversible. In contrast, DTNB, MTSEA, MTSES, and NPM had no effects on the current amplitude of P2X4S201C (Fig. 11, C and D), a mutant with a cysteine at 201 that stays slightly away from Trp-194, Arg-278, Asp-280, and Arg-282 (Figs. 11A and the lower panel of 2B). In addition, MTSEA, MTSES, DTNB, and NPM did not change the current amplitude of WT P2X4 (Fig. 11, C and D). Thus, specific covalent modifications at W194C are responsible for their inhibitory effects on the P2X4W194C.
      Figure thumbnail gr11
      Figure 11Weakening intersubunit physical coupling via covalent modifications. A, schematic structure of covalent modification on Cys-194 of P2X4W194C perturbs the H-bonds between Arg-278, Asp-280, and Arg-282 at the open state. B, reagents introduced additional charged or/and bulky groups into Cys-194 and Cys-201 of P2X4W194C and P2X4S201C, respectively. C and D, sample traces (C) and summarized (D, mean ± S.E., n = 3–5) effects of applications of MTSEA (1 mm), MTSES (1 mm), DTNB (1 mm), and NPM (1 mm) inhibited ATP-evoked currents of P2X4WT, P2X4S021C, or P2X4W194C, which were rescued following application of DTT except for NPM treatments. ∗∗, p ≪ 0.01 after versus before covalent modifications. Cells were voltage-clamped (amphotericin-perforated patch clamp) at −60 mV, and currents were evoked by ATP (100 μm, 15 s) at 8-min intervals. E and F, sample traces (E) and summarized (F, mean ± S.E., n = 3) effects of post MTSEA (1 mm) treatment on ATP (100 μm, saturated)-evoked remaining currents of P2X4WT, P2X4S021C, or P2X4W194C. ∗, p ≪ 0.05; ∗∗, p ≪ 0.01 after versus before MTSEA application, paired Student's t test. G and H, sample traces (G) and summarized effects (H) (mean ± S.E., n = 4–20) of mutations on the maximal amplitude of currents caused by saturated ATP (100 μm). ∗, p ≪ 0.05 versus WT (dashed line), one-way ANOVA with Bonferroni post hoc test.
      Similarly, covalent modification may also interrupt both conformations of the LF domain at resting and open states. To provide direct evidence that covalent modification could prevent the LF domain from fostering intersubunit physical couplings at the open state, MTSEA (1 mm) was applied after ATP to perturb the conformation of the N terminus of the LF domain at the open state. Post-administration of MTSEA inhibited 55 ± 7.2% of the remaining ATP currents of rP2X4W194C but only slightly or had no effects on rP2X4S201C and WT rP2X4 (Fig. 11, E and F, n = 3). Thus, the specific covalent modification-induced inhibitory effects may be due to perturbing the “interaction networks” among Arg-278, Asp-280, and Arg-282 at the open state, at least partially, which is essential for the formation of intersubunit physical couplings at the open state. Therefore, any conformational changes of the LF domain that interrupt the formation and stability of intersubunit physical couplings at the open state will affect the channel gating of P2X4 receptors.

      Discussion

      Here, we propose that the LF domain, a flexible loop structure, underwent allosteric changes after ATP binding, which promotes the formation of intersubunit physical couplings among the two lower body domains and the DF domain that facilitates opening of P2X4 receptors (Fig. 12). At the open state, bound ATP repels the LF domain out of the ATP-binding site, which coordinates the LF, DF, and two lower body domains into an integrated structural element. The integration of the LF domain in the cleft among those domains was achieved through the salt-bridge Arg-278…Asp-280, the newly formed hydrogen-bonding contacts Arg-282…Arg-278, Asn-192…Arg-282, Val-288…Arg-203, and Ser-289…Arg-203, and new hydrophobic interactions among Val-288, Ile-205, and Leu-214 after ATP binding (Fig. 12). The following evidence demonstrated that this new integrated structural element is stringently required by the channel gating of the P2X4 receptors. First, mutations of the residues involved in the establishment of physical couplings significantly reduced ATP currents of P2X4 receptors. The gel analysis has excluded the possibility that this newly integrated structural element made contributions to the channel stability and channel assembly. The absence of unitary current in R203A and R282A in response to saturated ATP and the “gain-of-function” of channel activity in these two mutants when IVM was co-applied with ATP confirmed the crucial role of intersubunit physical couplings fostered by the LF domain in the channel gating of P2X4 receptors. Covalent modifications on W194C-induced weakened channel activations of rP2X4 further supported this idea. Second, a slight alteration in the interdomain interactions among the LF, DF, and lower body domains, for example, introducing different engineered metal bridges between the LF and DF domains, switched the mutant rP2X4 receptors from “loss-of-function” (His-286/V288H) to “gain-of-function” (His-286/V288H/I209C) channels. Third, although alanine-screening mutagenesis revealed that the middle region of the LF domain exerts less influence in the channel activation, introducing intersubunit/interdomain disulfide cross-linking or metal bridge between the LF and lower body domains significantly affected the channel activation of rP2X4, suggesting that proper intersubunit/interdomain contacts, even in a region regarded as not so important, is still stringently required by the integrated structural element at the open state. Thus, bound ATP-induced repulsion of this flexible loop from the ATP-binding pocket is not a “passive”/inessential allosteric change that just accommodates itself to the allostery of the DF domain, but is an essential process that renders the establishment of new intersubunit physical couplings among the DF and two lower body domains, which integrates all those domains into a structural element that is stringently required by channel gating of rP2X4 receptors.
      Figure thumbnail gr12
      Figure 12Illustration of intersubunit physical couplings established by the LF domain at the open state. The established physical couplings integrate the DF, lower body, and LF domains into a structural element stringently required by the channel gating of rP2X4 receptors. The movements of extracellular domain and TM domain were referred by light-blue arrows. The black dotted lines connecting pink dots denote H-bonds or hydrophobic interactions between the key residues. Only two of three subunits, where subunit A and B are colored in green and purple, respectively, are shown for the clarity.
      Still, how the conformational changes of the LF domain together with the DF and two lower body domains affect the gating transition of P2X4 receptors needs to be explained. The deformed LF domain after ATP binding is sloped in a direction along the horizontal axis, making it possible to contact with both the rigid lower body and DF domains rather than the flexible loop β9-α3 (Fig. 6A), and thus possesses an ability in strengthening the physical couplings between the DF and two lower body domains (Fig. 12). Additionally, because mutations of W194A, W194E, W194R, E245A, and E245R (Figs. 4G and 11, G and H) and covalent modifications on S201C (Fig. 11, C–F) only produced small or no changes in the activation of the rP2X4, there should be a certain distance between the middle region of the LF domain of one subunit and the lower body domain of another subunit. This distance can avoid additional contacts between the middle region of the LF domain and the lower body domain during the outward moving of two lower body domains at the open state (Fig. 2, A and B). All of those features made the LF domain fit for coordinating both outward flexing of two domains and upward motion of the DF domain (Fig. 1, A and B). Because the DF domain is structurally coupled to the lower body domain through loop β9-α3 (Figs. 1, A and B, and 2, A–C), its upward motion evoked by ATP binding will cause the outward flexing of the lower body domains. Three lower body domains form the big central vestibule (
      • Hattori M.
      • Gouaux E.
      Molecular mechanism of ATP binding and ion channel activation in P2X receptors.
      ) of P2X receptors (Fig. 1, A and B), and the outward flexing of lower body domains leads to the expansion (
      • Hattori M.
      • Gouaux E.
      Molecular mechanism of ATP binding and ion channel activation in P2X receptors.
      ) of this big central vestibule (Fig. 1, A and B). It is worth noting that the expansion of the central vestibule is crucial for the channel gating of trimeric ion channels (
      • Baconguis I.
      • Hattori M.
      • Gouaux E.
      Unanticipated parallels in architecture and mechanism between ATP-gated P2X receptors and acid sensing ion channels.
      ,
      • Kellenberger S.
      • Grutter T.
      Architectural and functional similarities between trimeric ATP-gated P2X receptors and acid-sensing ion channels.
      ,
      • Rokic M.B.
      • Stojilkovic S.S.
      • Zemkova H.
      Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating.
      ), such as P2X receptors and ASIC channels. Small molecules (
      • Yu Y.
      • Chen Z.
      • Li W.G.
      • Cao H.
      • Feng E.G.
      • Yu F.
      • Liu H.
      • Jiang H.
      • Xu T.L.
      A nonproton ligand sensor in the acid-sensing ion channel.
      ,
      • Yu Y.
      • Li W.G.
      • Chen Z.
      • Cao H.
      • Yang H.
      • Jiang H.
      • Xu T.L.
      Atomic level characterization of the nonproton ligand-sensing domain of ASIC3 channels.
      ), toxins (
      • Baconguis I.
      • Bohlen C.J.
      • Goehring A.
      • Julius D.
      • Gouaux E.
      X-ray structure of acid-sensing ion channel 1-snake toxin complex reveals open state of a Na+-selective channel.
      ), and covalent modifications (
      • Yu Y.
      • Chen Z.
      • Li W.G.
      • Cao H.
      • Feng E.G.
      • Yu F.
      • Liu H.
      • Jiang H.
      • Xu T.L.
      A nonproton ligand sensor in the acid-sensing ion channel.
      ) acting on the residues of this region can directly affect the channel activation of ASIC channels. The outward flexing of the lower body domains and the expansion of the central vestibule might facilitate the channel activation of P2X receptors by the following reason. The central rigid lower body domains are structurally coupled with TM domains and the pore region (Fig. 1B). Therefore, the deflection of the LF domain evoked by bound ATP directly causes the motions of TM region through those rigid lower body domains, which may facilitate the gating transition from the resting state to the open state (Fig. 1A). Thus, the established physical couplings between the DF and two lower body domains by the deformed LF domain are pivotal to the outward flexing of lower body domains and the concomitant pore dilation of P2X4 receptors.
      Our data also showed that the flexible middle region of the LF domain, consisting of seemingly negligible residues, affected channel gating of P2X4 receptors. We have recently suggested that hydrophobic interactions between Val-288, Ile-205, Leu-214, and the aliphatic chain of Lys-190 in P2X4 receptors may develop an energy barrier for the channel gating (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ). As revealed by changes in both apparent affinity and maximal current before and after DTT application on disulfide cross-linking in mutant P2X4V288C/T211C, bound ATP-induced repelling action on Val-288 from the ATP-binding site may behave with two main functions. One is to reduce the energy barrier for channel gating, and the other is to accommodate ATP molecules. The N terminus of the LF domain is relative rigid because of its structural coupling with the lower body domain and the existence of a salt bridge between Arg-278 and Asp-280 in this region; thus, only a flexible middle region can buffer the repelling action of ATP on Val-288. Following the expulsion of Val-288 from the ATP-binding site and structural rearrangements of Val-288, Ile-205, Leu-214, and Lys-190, a lot of new intra- and intersubunit contacts were established, including Arg-282. At this stage, the middle region located on the interface between two “interaction clusters,” Asp-280…Arg-278…Arg-282 and Val-288…Arg-203…Ser-289, may act as a linker to stabilize those two “clusters” and maintains the intersubunit physical couplings between lower body domains and the DF domain at the open state. Thus, the middle region may contribute to the flexibility at the resting state to buffer the repelling action of ATP on Val-288 and provide the proper length between two termini (Fig. 10) that facilitate the LF domain to foster proper intersubunit physical couplings among the two lower body and DF domains at the open state.
      Finally, despite the indispensable role of the LF domain in channel activation of P2X4, we cannot neglect the fact that the sequence of the LF domain is not conserved among various subtypes of P2X receptors (Fig. 3D). At the open state, Arg-203 contacts with the main chain atoms of Val-288 and Ser-289, contributing to the establishment of intersubunit physical couplings. However, the arginine is replaced by glycine at the identical position of P2X2 and P2X3 subtypes (Fig. 3D). Similarly, the salt bridge formed by Arg-278 and Asp-280 in P2X4 is also absent in the P2X1 subtype (Fig. 3D). The identical residue of Arg-282 in P2X4 is absent in the P2X1 subtype (Fig. 3D). The sequence variation is much greater in the middle region of the LF domain throughout the P2X receptor family, which is completely absent in P2X6. However, this absence is not the only reason why no one could record ATP current in cells expressing P2X6 (
      • Jie Y.
      • Zhang L.
      • Xu H.
      • Gao C.
      • Ma W.
      • Li Z.
      Involvement of the left-flipper-to-dorsal-fin interface of the zebrafish P2X4 receptor in ATP binding and structural rearrangement.
      ). Because of those non-conserved sequences, the three-dimensional (3-D) architecture of the LF domain varies in different subtypes. Further studies are required to determine how those distinct sequences make up the functional LF domain in various subtypes. Nevertheless, it provides a foundation for developing subtype-specific blockers of P2X4 receptors by targeting on this non-conserved region of the LF domain throughout the P2X receptor family.
      In summary, ATP binding-induced repelling action on the LF domain promotes the formation of physical couplings among two lower body domains and the DF domain and facilitates the outward flexing of lower body domains (Fig. 12), leading to the expansion of the central vestibule and the concomitant pore dilation. This study provides new mechanistic insights into the channel gating of P2X4 receptors and may contribute to develop new strategies for subtype-specific blockers of P2X receptors.

      Experimental procedures

      Drugs, cell culture, mutagenesis, and receptor expression

      ATP, ZnCl2, and most of the other drugs were purchased from Sigma. The plasmid rP2X4 and zfP2X4.1 are the gifts from Drs. Lin-Hua Jiang, Alan North, and Eric Gouaux. Each mutant was constructed by the QuikChange mutagenesis kit and was verified by DNA sequencing. All constructs were expressed in cultured HEK-293 cells in DMEM at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Transfections of plasmids were performed using Hilymax (Dojindo Laboratories, Kumamoto, Japan). Electrophysiological measurements were performed on HEK-293 cells 24–48 h after transfection.

      Electrophysiology

      As in our previous descriptions (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ,
      • Yu Y.
      • Chen Z.
      • Li W.G.
      • Cao H.
      • Feng E.G.
      • Yu F.
      • Liu H.
      • Jiang H.
      • Xu T.L.
      A nonproton ligand sensor in the acid-sensing ion channel.
      ), conventional whole-cell configuration under the voltage clamp at room temperature (23 ± 2 °C) was used for electrophysiological recordings. Patch pipettes were pulled from glass capillaries using the two-stage puller PP-830 (Narishige Co., Ltd.), and the resistance between the recording electrode filled with pipette solution and the reference electrode in bath solution ranged from 3 to 5 megohms. Membrane currents were filtered at 2 kHz using a low pass Bessel filter and measured with an Axon 200B patch-clamp amplifier (Molecular Devices). All currents were sampled and analyzed in Digidata 1440 interface using Clampex and Clampfit 10.0 software (Molecular Devices). Cells were incubated in bath solution containing 150 mm NaCl, 5 mm KCl, 10 mm glucose, 10 mm HEPES, 2 mm CaCl2, and 1 mm MgCl2 at the conditional neutral pH 7.35–7.40. Patch electrodes were filled with standard internal solution containing 30 mm NaCl, 120 mm KCl, 1 mm MgCl2, 0.5 mm CaCl2, and 5 mm EGTA at the conditional neutral pH 7.35–7.40. During electrophysiological recordings, 80–90% of the series resistance was compensated, and the membrane potential was held at −60 mV throughout the experiment. As we described previously (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ), ATP solutions were prepared for 2 h in the batch buffer and applied using a fast pressure-driven computer-controlled microperfusion system OctaFlow08P (ALA Scientific Instrument). ATP currents were normalized to cell membrane capacitance. Dose-response curves data were collected from the recording of a range of ATP concentrations; the corresponding currents were normalized to the maximal current amplitude; ATP-gated currents were recorded after regular 3–5-s ATP application every 2–8 min. Pulses were spaced up to 8–20 min to avoid receptor desensitization at higher ATP concentration (10–100 μm) applications. The amphotericin-perforated patch-clamp technology (
      • Lewis C.J.
      • Evans R.J.
      Lack of run-down of smooth muscle P2X receptor currents recorded with the amphotericin permeabilized patch technique, physiological and pharmacological characterization of the properties of mesenteric artery P2X receptor ion channels.
      ) was also used for recordings of dose-dependent responses and covalent modifications of WT P2X4, P2X4W194C, and P2X4S201C. During this procedure, ATP-gated currents were recorded after regular 15–20-s ATP applications every 8–10 min to avoid receptor desensitization. Single-channel recordings using outside-out configuration were carried out in HEK-293 cells at room temperature (23 ± 2 °C) 24–48 h after transfection. Recording pipettes were pulled from borosilicate glass (World Precision Instruments, Inc.) and fire-polished to yield resistance of 5–10 megohms. The holding potential was −120 mV. The external solution and internal solutions are the same as those of whole-cell recordings. Single channel recordings were sampled at 50 kHz with a 2-kHz filter and a low-pass filtered at 200 Hz, using an AxoPatch 200B amplifier in conjunction with pClamp 10 software (Axon Instruments). Occasional large brief noise spikes were visually identified and removed from current traces.

      Cell-surface biotinylation and Western blotting analysis

      Cell-surface biotinylation and Western blotting were performed according to our previous descriptions (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ,
      • Yang Y.
      • Yu Y.
      • Cheng J.
      • Liu Y.
      • Liu D.S.
      • Wang J.
      • Zhu M.X.
      • Wang R.
      • Xu T.L.
      Highly conserved salt bridge stabilizes rigid signal patch at extracellular loop critical for surface expression of acid-sensing ion channels.
      ). Briefly, HEK-293 cells expressing rP2X4 or its mutants were washed in chilled PBS+/+ and then were incubated with sulfo-NHS-LC-biotin. The reaction was then terminated by treating the cells with glycine in PBS. Then the cells were collected and lysed with RIPA buffer. Using agarose resin linked to NeutrAvidin, the biotinylated proteins were then separated from the intracellular protein fraction. The resins were washed, and bound proteins were eluted with the boiling SDS sample buffer, whereas 10% of the volume of the supernatant was diluted and used as the total protein fraction. The samples were separated by SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane and then were incubated overnight at 4 °C with anti-EE tag (1:1000, Abcam, catalogue number ab40767) or anti-GAPDH (1:1000, Sungene Biotech, catalogue number KM9002) antibodies. Appropriate HRP-conjugated secondary antibodies for EE tag (25 °C, 1 h, 1:1000, goat-rabbit IgG(HL)-HRP; Sungene Biotech, catalogue number: LK2001) or GAPDH (25 °C, 1 h, 1:3000, goat-mouse IgG(HL)-HRP; Sungene Biotech, catalogue number: LK2003) were further incubated and finally visualized by exposure with the ImageQuant RT ECL system (GE Healthcare) for 1–3 min in ECL solution (Thermo Fisher Scientific). All Western blottings and gels are accompanied by the location of molecular weight markers (Thermo PageRuler Prestained Protein Ladder 10–170 kDa, catalogue number 26617). Protein expression analysis of each mutant and WT receptors was repeated at least by three independent experiments.

      Homology modeling

      For homology modeling of rP2X4 and its mutants using program MODELLER (
      • Sali A.
      • Blundell T.L.
      Comparative protein modelling by satisfaction of spatial restraints.
      ), the structures of the closed (PDB code 4DW0) and open (PDB code 4DW1) zfP2X4 receptors were taken as the templates. Zinc bridge models were also constructed according to our previous procedure (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ) using MODELLER. Briefly, for zinc-binding site reconstructions, a distance constraint (2.0–2.6 Å) was added between zinc and the coordination atoms of histidine or free cysteine residues. Then, these models applied by OPLS_2005 force field (
      • Jorgensen W.L.
      • Maxwell D.S.
      • Tirado-Rives J.
      Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids.
      ) were further minimized by DESMOND (
      • Shaw D.E.
      A fast, scalable method for the parallel evaluation of distance-limited pairwise particle interactions.
      ). The resulting models were further optimized by 1.2-ns MD simulations using program DESMOND with OPLS_2005 force field. After such a time scale of MD simulations, various parameters of zinc bridges, including distances between atoms, bond angles, and dihedral angles, were very close to those obtained by analysis of crystals (
      • Alberts I.L.
      • Nadassy K.
      • Wodak S.J.
      Analysis of zinc binding sites in protein crystal structures.
      ).

      MD simulations

      As we described previously (
      • Zhao W.S.
      • Wang J.
      • Ma X.J.
      • Yang Y.
      • Liu Y.
      • Huang L.D.
      • Fan Y.Z.
      • Cheng X.Y.
      • Chen H.Z.
      • Wang R.
      • Yu Y.
      Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation.
      ,
      • Yang H.
      • Yu Y.
      • Li W.G.
      • Yu F.
      • Cao H.
      • Xu T.L.
      • Jiang H.
      Inherent dynamics of the acid-sensing ion channel 1 correlates with the gating mechanism.
      ), all MD simulations were performed using the program DESMOND (
      • Shaw D.E.
      A fast, scalable method for the parallel evaluation of distance-limited pairwise particle interactions.
      ) with a constant number of particles, pressure, and temperature and periodic boundary conditions, which use a particular “neutral territory” method called the midpoint method (
      • Shaw D.E.
      A fast, scalable method for the parallel evaluation of distance-limited pairwise particle interactions.
      ) to efficiently exploit a high degree of computational parallelism. A default OPLS_2005 force field (
      • Jorgensen W.L.
      • Maxwell D.S.
      • Tirado-Rives J.
      Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids.
      ), following the functional form of the OPLS-AA family of force fields with additional stretch, bend, and torsional parameters for better coverage of ligand functional groups, was employed for the protein, ions, and ligand molecules. The energy-minimized homology models of rP2X4 and its mutants at the resting or open states were used as the starting structures for MD simulations. The large dimyristoylphosphatidylcholine bilayers in various simulation systems were constructed to generate a suitable membrane system where the TM region of the WT P2X4 and its mutants could be embedded. The protein/dimyristoylphosphatidylcholine system was then solvated in a bath of simple point charge water molecules. Counter ions were subsequently added to compensate for the net negative charge of the system. NaCl (150 mm) was added into the simulation box that represents background salt at physiological conditions. To maintain the system at a constant temperature of 300 K and constant pressure, Berendsen thermostat and barostat algorithms were applied to couple protein and other molecules. All of the bond lengths, including hydrogen atoms, were constrained by the Linear Constraint Solver algorithm. Electrostatic interactions between charged groups at a distance of less than 12 Å were calculated explicitly; long range electrostatic interactions were calculated using the smoothed particle mesh Ewald method. All of the MD simulations were run on the DAWNING TC2600 (AMD OpteronTM 8374HE CPUs). Preparation, analysis, and visualization were performed on a 12-CPU CORE DELL T7500 graphic working station. The MD trajectory analysis were performed using Simulation Even Analysis and Simulation Interactions Diagram tools of DESMOND.

      Metadynamics

      Metadynamics (
      • Laio A.
      • Rodriguez-Fortea A.
      • Gervasio F.L.
      • Ceccarelli M.
      • Parrinello M.
      Assessing the accuracy of metadynamics.
      ,
      • Limongelli V.
      • Bonomi M.
      • Marinelli L.
      • Gervasio F.L.
      • Cavalli A.
      • Novellino E.
      • Parrinello M.
      Molecular basis of cyclooxygenase enzymes (COXs) selective inhibition.
      • Laio A.
      • Gervasio F.L.
      Metadynamics: a method to simulate rare events and reconstruct the free energy in biophysics, chemistry and material science.
      ) is a technique where the potential for one or more chosen variables (“collective variables”) is modified by periodically adding a repulsive potential of Gaussian shape at the location given by particular values of the variables. All metadynamics analysis were conducted by the program DESMOND (
      • Shaw D.E.
      A fast, scalable method for the parallel evaluation of distance-limited pairwise particle interactions.
      ) under NPT and periodic boundary conditions using the default parameters at constant temperature (320 K) and pressure (1 bar) by using the Berendsen method. All simulations used the all-atom OPLS_2005 force field for proteins, ions, lipids, and the simple point charge waters. The parameters for height, width of the Gaussian, and the interval were set to 0.12 kcal/mol, 0.05 Å, and 0.09 ps, respectively. The sum of the Gaussians and the free-energy surface were generated by Metadynamics Analysis Tools of DESMOND.

      Data analysis

      The results are expressed as the means ± S.E. Statistical comparisons were made using one-way ANOVA and Student's t test, where p ≪ 0.05 (∗) or p ≪ 0.01 (∗∗) was considered significant. Concentration-response relationships for ATP activation of WT or mutated channels were obtained by measuring currents in response to different concentrations of ATP, and all of the results used to generate a concentration-response relationship were from the same group. The data were fit to Hill Equation 1,
      I/Imax=1/(1+(EC50/[ATP])n)
      (Eq. 1)


      where I is the normalized current at a given concentration of ATP; Imax is the maximum normalized current; EC50 is the concentration of ATP yielding a current that is half of the maximum, and n is the Hill coefficient.

      Author contributions

      Y. Yu designed the project. J. W., L. F. S., W. W. C., W. S. Z., X. F. M., B. L., and Y. M. H. performed cell culture, patch-clamp recording, and Western blotting. Y. L., J. W., Y. Yang, L. D. H., and Y. T. did mutations. Y. Yu and J. W. did MD simulations and metadynamics. J. W. and Y. Yu analyzed data. Y. Yu, X. Y. C., L. L., and X. Y. L. wrote the manuscript. All authors discussed the results and commented on the manuscript.

      Acknowledgments

      We thank Drs. Lin-Hua Jiang, Alan North, and Eric Gouaux for their kind gifts of plasmids rP2X4 and zfP2X4.1, and we thank Drs. Jing Yao and Yue-Zhou Li for their nice suggestions on the single channel recordings of rP2X4.

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