1. Structural Biology and Molecular Biophysics
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ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

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Cite this article as: eLife 2021;10:e64787 doi: 10.7554/eLife.64787

Abstract

Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through activated Panx1 channels that can support their many physiological roles.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files (Source Data files). Source data files have been provided for Figure 1G, Figure 1 Supplement 1C, Figure 1 Supplement 2B-E, Figure 2D, Figure 2 Supplement 1, Figure 2 Supplement 2A-F, Figure 3B-I, Figure 4 B-D, Figure 4 Supplement 2B-C. Source code has been uploaded to Github: https://github.com/VolkerKirchheim/VK_TIRFsinglevesicleStep1. Data is also available on Dryad under doi:10.5061/dryad.s1rn8pk69

The following data sets were generated

Article and author information

Author details

  1. Adishesh K Narahari

    Pharmacology, University of Virginia, Charlottesville, United States
    For correspondence
    akn4uq@virginia.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8708-9161
  2. Alex J B Kreutzberger

    Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9774-115X
  3. Pablo S Gaete

    Department of Pharmacology, Physiology & Neuroscience, New Jersey Medical School, Newark, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3373-9138
  4. Yu-Hsin Chiu

    Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4730-8104
  5. Susan A Leonhardt

    Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Christopher B Medina

    Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Xueyao Jin

    Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Patrycja W Oleniacz

    Pharmacology, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Volker Kiessling

    Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9388-5703
  10. Paula Q Barrett

    Pharmacology, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Kodi S Ravichandran

    Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Mark Yeager

    Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Jorge E Contreras

    Department of Pharmacology, Physiology & Neuroscience, New Jersey Medical School, Newark, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9203-1602
  14. Lukas K Tamm

    Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1674-4464
  15. Douglas Bayliss

    Pharmacology, University of Virginia, Charlottesville, United States
    For correspondence
    bayliss@virginia.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5630-2572

Funding

National Institutes of Health (P01 HL120840)

  • Kodi S Ravichandran
  • Mark Yeager
  • Douglas Bayliss

Ministry of Science and Technology, Taiwan (108-2320-B-007-007-MY2)

  • Yu-Hsin Chiu

National Institutes of Health (T32 GM007267)

  • Adishesh K Narahari

University of Virginia (Whitfield-Randolph Scholarship)

  • Adishesh K Narahari

National Institutes of Health (R01 HL138241)

  • Paula Q Barrett

National Institutes of Health (R01 GM099490)

  • Jorge E Contreras

National Institutes of Health (R01 HL48908)

  • Mark Yeager

National Institutes of Health (R01 GM138532)

  • Mark Yeager

National Institutes of Health (P01 GM072694)

  • Lukas K Tamm

National Institutes of Health (R01 GM051329)

  • Lukas K Tamm

National Institutes of Health (F30 CA236370)

  • Adishesh K Narahari

National Institutes of Health (T32 GM007055)

  • Adishesh K Narahari
  • Christopher B Medina

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Baron Chanda, Washington University in St. Louis, United States

Publication history

  1. Received: November 11, 2020
  2. Accepted: January 5, 2021
  3. Accepted Manuscript published: January 7, 2021 (version 1)
  4. Version of Record published: January 13, 2021 (version 2)

Copyright

© 2021, Narahari et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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