1. Biochemistry and Chemical Biology
  2. Neuroscience
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Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor

Research Article
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Cite this article as: eLife 2020;9:e57544 doi: 10.7554/eLife.57544

Abstract

Adenosine 5' triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase e subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

Article and author information

Author details

  1. Nami Kitajima

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9838-832X
  2. Kenji Takikawa

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
  3. Hiroshi Sekiya

    Department of Physiology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
  4. Kaname Satoh

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
  5. Daisuke Asanuma

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
  6. Hirokazu Sakamoto

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
  7. Shodai Takahashi

    Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
  8. Kenjiro Hanaoka

    Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0797-4038
  9. Yasuteru Urano

    Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
  10. Shigeyuki Namiki

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
  11. Masamitsu Iino

    Cellular and Molecular Pharmacology, Nihon University School of Medicine, Tokyo, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6426-4206
  12. Kenzo Hirose

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    For correspondence
    kenzoh@m.u-tokyo.ac.jp
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8944-6513

Funding

Ministry of Education, Culture, Sports, Science, and Technology (17H04029)

  • Kenzo Hirose

Ministry of Education, Culture, Sports, Science, and Technology (17K08584)

  • Shigeyuki Namiki

Japan Science and Technology Agency (JPMJPR17P1)

  • Daisuke Asanuma

Takeda Science Foundation

  • Nami Kitajima

Ministry of Education, Culture, Sports, Science, and Technology (19K22247)

  • Kenzo Hirose

Ministry of Education, Culture, Sports, Science, and Technology (25221304)

  • Masamitsu Iino

Ministry of Education, Culture, Sports, Science, and Technology (18K14915)

  • Hiroshi Sekiya

Ministry of Education, Culture, Sports, Science, and Technology (17H04764)

  • Daisuke Asanuma

Ministry of Education, Culture, Sports, Science, and Technology (18H04726)

  • Daisuke Asanuma

Ministry of Education, Culture, Sports, Science, and Technology (19K16251)

  • Hirokazu Sakamoto

Ministry of Education, Culture, Sports, Science, and Technology (18H04609)

  • Kenjiro Hanaoka

Ministry of Education, Culture, Sports, Science, and Technology (19H05414)

  • Kenjiro Hanaoka

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

Ethics

Animal experimentation: All procedures used in animal experiments were in accordance with the guidelines established by the Animal Welfare Committee of the University of Tokyo (Medicine-P10-010, Medicine-P15-017 and Medicine-P19-092).

Reviewing Editor

  1. Yamuna Krishnan, University of Chicago, United States

Publication history

  1. Received: April 3, 2020
  2. Accepted: July 9, 2020
  3. Accepted Manuscript published: July 10, 2020 (version 1)
  4. Version of Record published: August 3, 2020 (version 2)

Copyright

© 2020, Kitajima 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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