Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structures of human pannexin 1 reveal ion pathways and mechanism of gating


Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation1, apoptotic cell clearance2 and human oocyte development3. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Overall architecture of PANX1.
Fig. 2: CBX binding site of PANX1.
Fig. 3: A single PANX1 subunit.
Fig. 4: Channel assembly of PANX1.
Fig. 5: Ion-conducting pathways and channel gating.

Data availability

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-21588 (PANX1(WT)), EMD-21589 (PANX1(ΔCTT)), EMD-21590 (CBX–PANX(ΔCTT))00, EMD-21591 (PANX1(ΔNTH/ΔCTT)), EMD-21592 (CBX–PANX1(ΔNTH/ΔCTT)), EMD-21593 (PANX1(N255A)Hemi), EMD-21594 (PANX1(N255A)Gap), EMD-21595 (apo PANX1), EMD-21596 (Ca2+–PANX1), EMD-21597 (K+–PANX1) and EMD-21598 (SMA–PANX1). Structure models have been deposited in the RCSB Protein Data Bank under accession codes 6WBF (PANX1(WT)), 6WBG (PANX1(ΔCTT)), 6WBI (CBX–PANX(ΔCTT)), 6WBK (PANX1(ΔNTH/ΔCTT)), 6WBL (CBX–PANX1(ΔNTH/ΔCTT)), 6WBM (PANX1(N255A)Hemi) and 6WBN (PANX1(N255A)Gap).


  1. 1.

    Billaud, M. et al. A molecular signature in the pannexin1 intracellular loop confers channel activation by the α1 adrenoreceptor in smooth muscle cells. Sci. Signal. 8, ra17 (2015).

    PubMed  PubMed Central  CAS  Google Scholar 

  2. 2.

    Chekeni, F. B. et al. Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis. Nature 467, 863–867 (2010).

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  3. 3.

    Sang, Q. et al. A pannexin 1 channelopathy causes human oocyte death. Sci. Transl. Med. 11, eaav8731 (2019).

    PubMed  Google Scholar 

  4. 4.

    Tozaki-Saitoh, H., Tsuda, M. & Inoue, K. Role of purinergic receptors in CNS function and neuroprotection. Adv. Pharmacol. 61, 495–528 (2011).

    PubMed  CAS  Google Scholar 

  5. 5.

    Ren, J. & Bertrand, P. P. Purinergic receptors and synaptic transmission in enteric neurons. Purinergic Signal. 4, 255–266 (2008).

    PubMed  CAS  Google Scholar 

  6. 6.

    Tsuda, M., Tozaki-Saitoh, H. & Inoue, K. Pain and purinergic signaling. Brain Res. Rev. 63, 222–232 (2010).

    PubMed  CAS  Google Scholar 

  7. 7.

    Le, T.-T. T. et al. Purinergic signaling in pulmonary inflammation. Front. Immunol. 10, 1633 (2019).

    PubMed  PubMed Central  CAS  Google Scholar 

  8. 8.

    Zhang, Z. et al. Regulated ATP release from astrocytes through lysosome exocytosis. Nat. Cell Biol. 9, 945–953 (2007).

    PubMed  CAS  Google Scholar 

  9. 9.

    Dahl, G. ATP release through pannexon channels. Phil. Trans. R. Soc. Lond. B 370, 20140191 (2015).

    Google Scholar 

  10. 10.

    Taruno, A. et al. CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes. Nature 495, 223–226 (2013).

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  11. 11.

    Anselmi, F. et al. ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear. Proc. Natl Acad. Sci. USA 105, 18770–18775 (2008).

    ADS  PubMed  CAS  Google Scholar 

  12. 12.

    Penuela, S., Gehi, R. & Laird, D. W. The biochemistry and function of pannexin channels. Biochim. Biophys. Acta 1828, 15–22 (2013).

    PubMed  CAS  Google Scholar 

  13. 13.

    Adamson, S. E. et al. Pannexin 1 is required for full activation of insulin-stimulated glucose uptake in adipocytes. Mol. Metab. 4, 610–618 (2015).

    PubMed  PubMed Central  CAS  Google Scholar 

  14. 14.

    Makarenkova, H. P., Shah, S. B. & Shestopalov, V. I. The two faces of pannexins: new roles in inflammation and repair. J. Inflamm. Res. 11, 273–288 (2018).

    PubMed  PubMed Central  CAS  Google Scholar 

  15. 15.

    Thompson, R. J. et al. Activation of pannexin-1 hemichannels augments aberrant bursting in the hippocampus. Science 322, 1555–1559 (2008).

    ADS  PubMed  CAS  Google Scholar 

  16. 16.

    Silverman, W. R. et al. The pannexin 1 channel activates the inflammasome in neurons and astrocytes. J. Biol. Chem. 284, 18143–18151 (2009).

    PubMed  PubMed Central  CAS  Google Scholar 

  17. 17.

    Crespo Yanguas, S. et al. Pannexin1 as mediator of inflammation and cell death. Biochim. Biophys. Acta 1864, 51–61 (2017).

    CAS  Google Scholar 

  18. 18.

    Michalski, K. & Kawate, T. Carbenoxolone inhibits pannexin1 channels through interactions in the first extracellular loop. J. Gen. Physiol. 147, 165–174 (2016).

    PubMed  PubMed Central  CAS  Google Scholar 

  19. 19.

    Poon, I. K. H. et al. Unexpected link between an antibiotic, pannexin channels and apoptosis. Nature 507, 329–334 (2014).

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  20. 20.

    Myers, J. B. et al. Structure of native lens connexin 46/50 intercellular channels by cryo-EM. Nature 564, 372–377 (2018).

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  21. 21.

    Maeda, S. et al. Structure of the connexin 26 gap junction channel at 3.5 Å resolution. Nature 458, 597–602 (2009).

    ADS  PubMed  CAS  Google Scholar 

  22. 22.

    Oshima, A., Tani, K. & Fujiyoshi, Y. Atomic structure of the innexin-6 gap junction channel determined by cryo-EM. Nat. Commun. 7, 13681 (2016).

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  23. 23.

    Kefauver, J. M. et al. Structure of the human volume regulated anion channel. eLife 7, e38461 (2018).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Kasuya, G. et al. Cryo-EM structures of the human volume-regulated anion channel LRRC8. Nat. Struct. Mol. Biol. 25, 797–804 (2018).

    PubMed  CAS  Google Scholar 

  25. 25.

    Deneka, D., Sawicka, M., Lam, A. K. M., Paulino, C. & Dutzler, R. Structure of a volume-regulated anion channel of the LRRC8 family. Nature 558, 254–259 (2018).

    ADS  PubMed  CAS  Google Scholar 

  26. 26.

    Kern, D. M., Oh, S., Hite, R. K. & Brohawn, S. G. Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs. eLife 8, e42636 (2019).

    PubMed  PubMed Central  Google Scholar 

  27. 27.

    Choi, W., Clemente, N., Sun, W., Du, J. & Lü, W. The structures and gating mechanism of human calcium homeostasis modulator 2. Nature 576, 163–167 (2019).

    ADS  PubMed  CAS  Google Scholar 

  28. 28.

    Syrjanen, J. L. et al. Structure and assembly of calcium homeostasis modulator proteins. Nat. Struct. Mol. Biol. 27, 150–159 (2020).

    PubMed  PubMed Central  CAS  Google Scholar 

  29. 29.

    Sosinsky, G. E. et al. Pannexin channels are not gap junction hemichannels. Channels (Austin) 5, 193–197 (2011).

    CAS  Google Scholar 

  30. 30.

    Boassa, D. et al. Pannexin1 channels contain a glycosylation site that targets the hexamer to the plasma membrane. J. Biol. Chem. 282, 31733–31743 (2007).

    PubMed  CAS  Google Scholar 

  31. 31.

    Beckmann, A., Grissmer, A., Krause, E., Tschernig, T. & Meier, C. Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells. Cell Tissue Res. 363, 751–763 (2016).

    PubMed  CAS  Google Scholar 

  32. 32.

    Sahu, G., Sukumaran, S. & Bera, A. K. Pannexins form gap junctions with electrophysiological and pharmacological properties distinct from connexins. Sci. Rep. 4, 4955 (2014).

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  33. 33.

    Sandilos, J. K. et al. Pannexin 1, an ATP release channel, is activated by caspase cleavage of its pore-associated C-terminal autoinhibitory region. J. Biol. Chem. 287, 11303–11311 (2012).

    PubMed  PubMed Central  CAS  Google Scholar 

  34. 34.

    Chiu, Y.-H. et al. A quantized mechanism for activation of pannexin channels. Nat. Commun. 8, 14324 (2017).

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  35. 35.

    Michalski, K., Henze, E., Nguyen, P., Lynch, P. & Kawate, T. The weak voltage dependence of pannexin 1 channels can be tuned by N-terminal modifications. J. Gen. Physiol. 150, 1758–1768 (2018).

    PubMed  PubMed Central  CAS  Google Scholar 

  36. 36.

    Wang, J. et al. The membrane protein pannexin1 forms two open-channel conformations depending on the mode of activation. Sci. Signal. 7, ra69 (2014).

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Locovei, S., Wang, J. & Dahl, G. Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium. FEBS Lett. 580, 239–244 (2006).

    PubMed  CAS  Google Scholar 

  38. 38.

    DeLalio, L. J. et al. Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane. J. Biol. Chem. 294, 6940–6956 (2019).

    PubMed  PubMed Central  CAS  Google Scholar 

  39. 39.

    Weilinger, N. L. et al. Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity. Nat. Neurosci. 19, 432–442 (2016).

    PubMed  CAS  Google Scholar 

  40. 40.

    Furlow, P. W. et al. Mechanosensitive pannexin-1 channels mediate microvascular metastatic cell survival. Nat. Cell Biol. 17, 943–952 (2015).

    PubMed  PubMed Central  CAS  Google Scholar 

  41. 41.

    Penuela, S., Celetti, S. J., Bhalla, R., Shao, Q. & Laird, D. W. Diverse subcellular distribution profiles of pannexin 1 and pannexin 3. Cell Commun. Adhes. 15, 133–142 (2008).

    PubMed  CAS  Google Scholar 

  42. 42.

    Michalski, K. et al. The cryo-EM structure of pannexin 1 reveals unique motifs for ion selection and inhibition. eLife 9, e54670 (2020).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Deng, Z. et al. Cryo-EM structures of the ATP release channel pannexin 1. Preprint at (2020).

  44. 44.

    Ma, W. et al. Pannexin 1 forms an anion-selective channel. Pflugers Arch. 463, 585–592 (2012).

    PubMed  CAS  Google Scholar 

  45. 45.

    Wang, J. & Dahl, G. SCAM analysis of Panx1 suggests a peculiar pore structure. J. Gen. Physiol. 136, 515–527 (2010).

    PubMed  PubMed Central  CAS  Google Scholar 

  46. 46.

    Romanov, R. A. et al. The ATP permeability of pannexin 1 channels in a heterologous system and in mammalian taste cells is dispensable. J. Cell Sci. 125, 5514–5523 (2012).

    PubMed  PubMed Central  CAS  Google Scholar 

  47. 47.

    Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).

    PubMed  PubMed Central  CAS  Google Scholar 

  48. 48.

    Zhou, Q. et al. Target protease specificity of the viral serpin CrmA. Analysis of five caspases. J. Biol. Chem. 272, 7797–7800 (1997).

    PubMed  CAS  Google Scholar 

  49. 49.

    Denault, J.-B. & Salvesen, G. S. Expression, purification, and characterization of caspases. Curr. Protoc. Protein Sci. 30, 21.13.1– 21.13.15 (2002).

    Google Scholar 

  50. 50.

    Haley, E. et al. Expression and purification of the human lipid-sensitive cation channel TRPC3 for structural determination by single-particle cryo-electron microscopy. J. Vis. Exp. 143, e58754 (2019).

    Google Scholar 

  51. 51.

    Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    PubMed  Google Scholar 

  52. 52.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    PubMed  PubMed Central  CAS  Google Scholar 

  53. 53.

    Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  54. 54.

    Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    PubMed  PubMed Central  Google Scholar 

  55. 55.

    Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    PubMed  PubMed Central  CAS  Google Scholar 

  56. 56.

    Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    PubMed  PubMed Central  CAS  Google Scholar 

  57. 57.

    Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019).

    PubMed  PubMed Central  CAS  Google Scholar 

  58. 58.

    Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ 7, 253–267 (2020).

    PubMed  PubMed Central  CAS  Google Scholar 

  59. 59.

    Seminario-Vidal, L. et al. Thrombin promotes release of ATP from lung epithelial cells through coordinated activation of Rho- and Ca2+-dependent signaling pathways. J. Biol. Chem. 284, 20638–20648 (2009).

    PubMed  PubMed Central  CAS  Google Scholar 

  60. 60.

    Chiu, Y.-H., Schappe, M. S., Desai, B. N. & Bayliss, D. A. Revisiting multimodal activation and channel properties of pannexin 1. J. Gen. Physiol. 150, 19–39 (2018).

    PubMed  PubMed Central  CAS  Google Scholar 

  61. 61.

    Nielsen, B. S. et al. Pannexin 1 activation and inhibition is permeant-selective. J. Physiol. 598, 361–379 (2020).

    PubMed  CAS  Google Scholar 

  62. 62.

    Dourado, M., Wong, E. & Hackos, D. H. Pannexin-1 is blocked by its C-terminus through a delocalized non-specific interaction surface. PLoS ONE 9, e99596 (2014).

    ADS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Penuela, S., Bhalla, R., Nag, K. & Laird, D. W. Glycosylation regulates pannexin intermixing and cellular localization. Mol. Biol. Cell 20, 4313–4323 (2009).

    PubMed  PubMed Central  CAS  Google Scholar 

  64. 64.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    PubMed  PubMed Central  Google Scholar 

  65. 65.

    Drozdetskiy, A., Cole, C., Procter, J. & Barton, G. J. JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 43, W389–W394 (2015).

    PubMed  PubMed Central  CAS  Google Scholar 

  66. 66.

    Afonine, P. V. et al. New tools for the analysis and validation of cryo-EM maps and atomic models. Acta Crystallogr. D 74, 814–840 (2018).

    CAS  Google Scholar 

  67. 67.

    Trabuco, L. G., Villa, E., Schreiner, E., Harrison, C. B. & Schulten, K. Molecular dynamics flexible fitting: a practical guide to combine cryo-electron microscopy and X-ray crystallography. Methods 49, 174–180 (2009).

    PubMed  PubMed Central  CAS  Google Scholar 

  68. 68.

    Williams, C. J. et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    PubMed  PubMed Central  CAS  Google Scholar 

  69. 69.

    The PyMOL Molecular Graphics System v.2.1. (Schrödinger, 2020).

  70. 70.

    Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001).

    ADS  PubMed  CAS  Google Scholar 

  71. 71.

    Chovancova, E. et al. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLOS Comput. Biol. 8, e1002708 (2012).

    PubMed  PubMed Central  CAS  Google Scholar 

  72. 72.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    PubMed  CAS  Google Scholar 

  73. 73.

    Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    PubMed  CAS  Google Scholar 

  74. 74.

    Shen, M. R. et al. Differential expression of volume-regulated anion channels during cell cycle progression of human cervical cancer cells. J. Physiol. 529, 385–394 (2000).

    PubMed  PubMed Central  CAS  Google Scholar 

  75. 75.

    Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).

    PubMed  CAS  Google Scholar 

  76. 76.

    Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I. & Lomize, A. L. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res. 40, D370–D376 (2012).

    PubMed  CAS  Google Scholar 

  77. 77.

    Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat. Methods 14, 71–73 (2017).

    PubMed  CAS  Google Scholar 

  78. 78.

    Hess, B. P-LINCS: a parallel linear constraint solver for molecular simulation. J. Chem. Theory Comput. 4, 116–122 (2008).

    PubMed  CAS  Google Scholar 

  79. 79.

    Abraham, M. J. et al. Gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).

    ADS  Google Scholar 

Download references


We thank B. Roth for the initial construct screening; G. Zhao and X. Meng for the support with data collection at the David Van Andel Advanced Cryo-Electron Microscopy Suite; the HPC team of VARI for computational support; and D. Nadziejka for technical editing. W.L. is supported by the National Institutes of Health (NIH) (grant R56HL144929). J.D. is supported by a McKnight Scholar Award, a Klingenstein-Simon Scholar Award, a Sloan Research Fellowship in neuroscience and the NIH (grant R01NS111031). Z.R. is supported by an American Heart Association postdoctoral fellowship (grant 20POST35120556).

Author information




W.L. and J.D. initiated and supervised the project. Z.R. performed mutagenesis, purified PANX1, prepared and screened cryo-EM samples and performed cryo-EM data collection and processing and computational simulation. I.J.O. performed electrophysiological experiments. All authors contributed in manuscript preparation.

Corresponding authors

Correspondence to Juan Du or Wei Lü.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Stephen Brohawn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Purification and biochemical analysis of PANX1.

a, SEC profile of PANX1 purification using GDN. b, SDS–PAGE of purified PANX1–GFP. For gel source data, see Supplementary Fig. 1a. c, FSEC experiment for caspase 3/7 cleavage of PANX1–GFP. GFP absorbance (480 nm) is shown on the y-axis. d, Caspase 7 cleavage of purified PANX1–GFP. Cleavage of CTT results in a peak shift. Tryptophan absorbance (280 nm) is shown on the y-axis. e, SMA solubilization screening of PANX1–GFP using FSEC. Three SMA polymers (SMA25010, SMA30010 and SMA40001) were tested. GFP absorbance (480 nm) is shown on the y-axis. f, SEC profile of PANX1 purification using SMA30010. Tryptophan absorbance (280 nm) is shown on the y-axis. g, Deglycosylation test of PANX1–GFP and PANX1(N255A)–GFP using PNGase F. Bands corresponding to the glycosylated and non-glycosylated PANX1 are indicated. See Supplementary Fig. 1b for gel source data. h, FSEC analysis of the PANX1 mutations used for electrophysiology recordings. Cells expressing PANX1(WT) or mutants were solubilized using GDN. Gain-of-function mutations with lower expression level are labelled. The R75E mutant contains a peak position shifted to the right. i, j, FSEC analysis on extracellular-gate mutations of PANX1 solubilized using GDN (i) or SMA30010 (j). Peak positions of correctly assembled PANX1 and incorrectly assembled PANX1 are indicated by arrows and vertical bars. The W74R, R75D and R75E mutants showed decreased stability relative to wild type because, when extracted using detergent, they mostly ran at positions representing incorrect assemblies (i). Nevertheless, the SMA-extracted W74R, R75D and R75E mutants still showed peaks at positions representing correct assemblies (j), indicating that they are able to form correctly assembled channel complex in a native lipid environment.

Extended Data Fig. 2 The workflow of cryo-EM data processing of PANX1 constructs that do not form gap junctions.

a, The data-analysis pipeline for PANX1 cryo-EM analysis with no gap junction. Special attention was paid to determine whether the dataset indeed adheres to C7 symmetry. Two examples of PANX1 adopting a symmetric conformation (PANX1(ΔCTT)) or a non-symmetric conformation (CBX–PANX1(ΔCTT)) are shown. A detailed description of the data-analysis procedure can be found in Methods. b, The overlay of the C1 refined maps of PANX1(ΔCTT), CBX–PANX1(ΔCTT), PANX1(ΔNTH/ΔCTT) and CBX–PANX1(ΔNTH/ΔCTT) with the symmetric model of PANX1. The CBX–PANX1(ΔCTT), PANX1((ΔNTH/ΔCTT)) and CBX–PANX1(ΔNTH/ΔCTT) maps adopt a non-symmetric shape.

Extended Data Fig. 3 Representative micrographs, 2D class averages and Fourier shell correlation curves for all datasets in this study.

aj, For each dataset, a representative micrograph, four 2D class averages and Fourier shell correlation (FSC) curve plot are shown, except for PANX1(N255A) dataset, in which two structures are shown. The map resolution is determined on the basis of the gold-standard 0.143 criterion. If an atomic model is available for the dataset, a model versus map FSC curve is also provided. The model versus map resolution is determined on the basis of the 0.5 FSC criterion. In a, a slice view of the SMA–PANX1 map showing the organization of the TMD is shown.

Extended Data Fig. 4 Local resolution estimation and representative densities.

a, b, PANX1 map. c, d, The PANX1(N255A)Gap map. To provide better visualization on the exterior and interior map quality, both non-sliced and sliced views of the maps are shown. The colour represents the local resolution in Å. Representative densities are shown for a few selected secondary structure elements of PANX1.

Extended Data Fig. 5 The workflow of cryo-EM data processing for PANX1(N255A).

To separate gap junction particles from hemichannel particles, we relied on 2D classification to distinguish tilted and side views. The top and down views were separated during 3D classification. A more detailed description of the data-analysis pipeline can be found in the Methods.

Extended Data Fig. 6 The structures of PANX1(ΔNTH/ΔCTT).

a, The apo state. b, PANX1(ΔNTH/ΔCTT) in complex with CBX. CBX is shown in orange. In a, b, odd- and even-numbered subunits are shown in blue and white, respectively; the seventh subunit is in green. Cryo-EM maps viewed parallel to the membrane (left) and from the extracellular side (middle). The unsharpened map is shown as a transparent envelope. Right, structural models viewed from the intracellular side. c, The slice view of the extracellular entrance of PANX1(ΔNTH/ΔCTT) (left) and CBX–PANX1(ΔNTH/ΔCTT) (right) maps. The CBX molecule and the side chain of W74 are shown in stick.

Extended Data Fig. 7 Patch-clamp analysis of the wild-type PANX1 and its mutants.

a, Representative traces of whole-cell current density from patch-clamped tsA201 control cells (n = 6) and tsA201 cells overexpressing: wild type (n = 12) and ΔNTH (n = 5), W74A (n = 8), R75E (n = 5), N255A (n = 5), Δ21–23 (n = 6), Δ21–27 (n = 7), R29A (n = 9), A33W (n = 10), A33W/ΔCTT (n = 4), ΔCTT (n = 4) and A33C (n = 5) mutant PANX1. Voltage steps (0.25 s) of 20 mV were imposed from −100 mV to +80 mV from a holding potential of −10 mV. Cells were first measured in standard bath solution and then re-measured following the superfusion of a bath solution containing 0.1 mM carbenoxolone. b, Mean current measured at 5 ms of experiments in a plotted as a function of clamp voltage. c, Current amplitudes of experiments in a with and without CBX. Each paired point represents an individual cell and the bar represents the mean. d, Plot of zero-current reversal potentials using a 2-s voltage ramp for various bath solutions containing either (in mM): 145 NaCl, 145 NaI, 145 sodium gluconate, 14.5 NaCl or 145 NMDG-Cl (see Methods for complete solutions). The number of cells patched (indicated in parenthesis) for the various bath solutions were as follows, 145 mM NaCl: wild type (5), R29A (7), W74A (5), R75E (7); 145 mM NaI: wild type (5), R29A (7), W74A (5), R75E (7); 145 mM sodium gluconate: wild type (5), R29A (7), W74A (5), R75E (7); 14.5 mM NaCl: wild type (4), R29A (4), W74A (5), R75E (5); 145 mM NMDG-Cl: wild type (3), R29A (2), W74A (5), R75E (3). e, f, Plots of the calculated (Methods) permeability of iodide relative to chloride (PI/PCl), the permeability of gluconate relative to chloride (Pgluconate/PCl) and permeability of sodium relative to chloride (PNa/PCl) for wild-type and mutant channels. For statistical comparisons to wild type, one-way analysis of variance with Bonferroni correction was performed. For PI/PCl, P = 0.99 (R29A), 3.7 × 10−5 (W74A) and 5.9 × 10−5 (R75E). For Pgluconate/PCl, P = 0.046 (R29A), 0.99 (W74A) and 6.8 × 10−9 (R75E). For PNa/PCl, P = 0.99 (R29A), 0.99 (W74A) and 2.2 × 10−6 (R75E). Each point represents an individual cell. The bar represents mean and error bars show s.e.m.

Extended Data Fig. 8 Comparison of large-pore channels.

a, The structures of large-pore channels, viewed parallel (top) or perpendicular (bottom) to the membrane. One subunit (or one pair of subunits) is in green. The diameter of VRAC is calculated without the cytoplasmic leucine-rich repeat domain. b, Organization of the TMD, viewed from the intracellular side. The NTH and transmembrane helices S1, S2, S3 and S4 are labelled for two subunits. Only CALHM2 has its transmembrane helices arranged in a clockwise manner. The contact between adjacent TMDs in PANX1 is made by the NTH with the S1 and S2 helices in the neighbouring subunit; the same contact in CALHM2 is made by the S2 and S4 of adjacent subunits, and in connexin by the S1 and S2 of adjacent subunits. There is no major contact in innexin and VRAC.

Extended Data Fig. 9 Secondary structure arrangement and sequence alignment.

Secondary structures based on the PANX1 structure model are labelled. The W74 forming the extracellular entrance is marked with an arrow. Key residues forming the side tunnel are labelled with a red asterisk. The cysteine residues forming the extracellular disulfide bonds are highlighted by an orange dot. The N255 glycosylation site is marked with a green dot. The gap junction interface and caspase 3/7 cleavage site are indicated with a red frame. A gain-of-function disease mutation (Δ21–23) of PANX1 is also marked.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics for PANX1(WT), apo PANX1(WT), Ca2+–PANX1(WT) and K+–PANX1(WT)
Extended Data Table 2 Cryo-EM data collection, refinement and validation statistics for PANX1(ΔCTT), CBX–PANX1(ΔCTT), PANX1(ΔNTH/ΔCTT) and CBX–PANX1(ΔNTH/ΔCTT)
Extended Data Table 3 Cryo-EM data collection, refinement and validation statistics for PANX1(N255A)Hemi, PANX1(N255A)Gap, SMA–PANX1(WT)

Supplementary information

Supplementary Figure 1

This file contains the raw gel images for Extended Data Fig. 1b and 1g.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ruan, Z., Orozco, I.J., Du, J. et al. Structures of human pannexin 1 reveal ion pathways and mechanism of gating. Nature 584, 646–651 (2020).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing