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Cryo-EM structures of the ATP release channel pannexin 1

Nature Structural & Molecular Biology volume 27, pages 373–381 (2020)Cite this article

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Abstract

The plasma membrane adenosine triphosphate (ATP) release channel pannexin 1 (PANX1) has been implicated in many physiological and pathophysiological processes associated with purinergic signaling, including cancer progression, apoptotic cell clearance, inflammation, blood pressure regulation, oocyte development, epilepsy and neuropathic pain. Here we present near-atomic-resolution structures of human and frog PANX1 determined by cryo-electron microscopy that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the size of permeable substrates. The amino and carboxyl termini, not resolved in the density map, appeared to be structurally dynamic and might contribute to narrowing of the pore during channel gating. In combination with functional characterization, this work elucidates the previously unknown architecture of pannexin channels and establishes a foundation for understanding their unique channel properties.

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Fig. 1: Electrophysiology and cryo-EM reconstruction of xPANX1.
Fig. 2: Structure of xPANX1.
Fig. 3: Pore architecture and electrostatic properties.
Fig. 4: Comparison of cryo-EM reconstructions of human and frog PANX1.
Fig. 5: Inter-subunit interface.
Fig. 6: Structural comparison of the extended 4-TM family of channels.

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Data availability

The cryo-EM maps of hPANX1 and xPANX1 have been deposited in the Electron Microscopy Data Bank with accession codes EMD-21071 and EMD-20964. Atomic coordinates for hPANX1 and xPANX1 structures have been deposited in the Protein Data Bank with accession codes 6V6D and 6UZY. Source data for Fig. 1a–d and Extended Data Figs. 2a, 4a and 5 are available with the paper online.

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Acknowledgements

This work was supported by startup funds from the Washington University School of Medicine (to P.Y.). M.J.R and J.A.J.F are supported by the Washington University Center for Cellular Imaging, which is funded in part by the Washington University School of Medicine through the Precision Medicine Initiative, the Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (CDI-CORE-2015-505 and CDI-CORE-2019-813) and the Foundation for Barnes-Jewish Hospital (3770).

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Author notes

  1. These authors contributed equally: Zengqin Deng, Zhihui He.

Authors and Affiliations

  1. Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA

    Zengqin Deng, Zhihui He, Grigory Maksaev, Ryan M. Bitter, James A. J. Fitzpatrick & Peng Yuan

  2. Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, St. Louis, MO, USA

    Zengqin Deng, Zhihui He, Grigory Maksaev, Ryan M. Bitter & Peng Yuan

  3. Washington University Center for Cellular Imaging, Washington University School of Medicine, St. Louis, MO, USA

    Michael Rau & James A. J. Fitzpatrick

  4. Department of Neuroscience, Washington University School of Medicine, St. Louis, MO, USA

    James A. J. Fitzpatrick

  5. Department of Biomedical Engineering, Washington University in Saint Louis, St. Louis, MO, USA

    James A. J. Fitzpatrick

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Contributions

Z.D., Z.H. and R.M.B. performed biochemical preparations, cryo-EM data processing, structural determination and analysis. G.M. conducted electrophysiology experiments. M.R., J.A.J.F., Z.D. and Z.H. collected cryo-EM data. Z.H. performed the crosslinking experiments. P.Y. designed and supervised the project. Z.D., Z.H., G.M. and P.Y. analyzed the results and prepared the manuscript with input from all authors. Correspondence and requests for materials should be addressed to P.Y.

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Correspondence to Peng Yuan.

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Extended data

Extended Data Fig. 1 CBX inhibition of the wild-type xPANX1 and mutants in excised inside-out membrane patches.

a,b, Current-voltage relationship of wild-type xPANX1 in symmetrical NaCl (a) and KCl (b) in the absence (black) and presence (gray) of 100 µM CBX. c-e, Current-voltage relationship of the W74A (c), R75A (d), and D81A (e) mutants in symmetrical NaCl in the absence (black) and presence (gray) of 100 µM CBX.

Extended Data Fig. 2 Cryo-EM reconstruction of xPANX1.

a, Purification of xPANX1 on size-exclusion chromatography. Fractions of the monodisperse channel peak were shown in SDS-PAGE (inset). The uncropped gel image is available as source data. b, Flowchart of cryo-EM image processing. c, Fourier shell correlation before and after post-processing in RELION3. d, Fourier shell correlation between the refined model and the full map. e, Cryo-EM density map colored by local resolution in the range of 3.0-4.0 Å. f, Angular distribution plot of particles used in the final reconstruction. Only one-seventh of the sphere is shown owing to applied C7 symmetry.

Source data

Extended Data Fig. 3 Cryo-EM density map of xPANX1.

Cryo-EM density for an entire channel subunit as well as for selected regions is shown as blue mesh. Residues forming disulfide bonds are highlighted.

Extended Data Fig. 4 Cryo-EM reconstruction of human PANX1.

a, Purification of hPANX1 on size-exclusion chromatography. Fractions of the monodisperse channel peak were shown in SDS-PAGE (inset) and protein samples used for cryo-EM were indicated. The uncropped gel image is available as source data. b, Flowchart of cryo-EM image processing. c, Fourier shell correlation before and after post-processing in RELION3. d, Fourier shell correlation between the refined model and the full map. e, Cryo-EM density map colored by local resolution in the range of 3.5-6.0 Å. f, Angular distribution plot of particles used in the final reconstruction.

Source data

Extended Data Fig. 5 Crosslinking of PANX1 channels.

a, SDS-PAGE showing crosslinking of purified hPANX1 and xPANX1 proteins expressed in yeast P. pastoris. A ladder of seven distinct oligomers was detected at a medium concentration of crosslinking reagent disuccinimidyl suberate (DSS, 250 μM). At high concentrations of DSS, both xPANX1 and hPANX1 were primarily crosslinked to heptamers. Numbers on the right indicate numbers of crosslinked subunits. b, Western blots showing crosslinking of hPANX1 in the plasma membranes of mammalian cells (HEK293S). With 75 μM DSS, a ladder of crosslinked oligomers up to heptamer was observed. At high concentrations of DSS, hPANX1 protein is crosslinked to heptamers and even larger size assemblies. Uncropped images are available as source data.

Source data

Supplementary information

Source data

Source Data Fig. 1

Statistical source data

Source Data Extended Data Fig. 2

Full-length unprocessed SDS-PAGE gel

Source Data Extended Data Fig. 4

Full-length unprocessed SDS-PAGE gel

Source Data Extended Data Fig. 5

Full-length unprocessed SDS-PAGE gel and western blots

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Deng, Z., He, Z., Maksaev, G. et al. Cryo-EM structures of the ATP release channel pannexin 1. Nat Struct Mol Biol 27, 373–381 (2020). https://doi.org/10.1038/s41594-020-0401-0

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