Improved ANAP incorporation and VCF analysis reveals details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain

  1. Anna Durner
  2. Ellis Durner
  3. Annette Nicke  Is a corresponding author
  1. LMU Munich, Germany

Abstract

The large intracellular C-terminus of the pro-inflammatory P2X7 ion channel receptor (P2X7R) is associated with diverse P2X7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2X7R recently identified a membrane-associated anchoring domain, an open-state stabilizing 'cap' domain, and a globular 'ballast domain' containing GTP/GDP and dinuclear Zn2+-binding sites with unknown functions. To investigate protein dynamics during channel activation, we improved incorporation of the environment-sensitive fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) into Xenopus laevis oocyte-expressed P2X7Rs and performed voltage clamp fluorometry (VCF). While we confirmed predicted conformational changes within the extracellular and the transmembrane domains, only three out of 41 mutants containing ANAP in the C-terminal domain resulted in ATP-induced fluorescence changes. We conclude that the ballast domain functions rather independently from the extracellular ATP binding domain and might require activation by additional ligands and/or protein interactions. Novel tools to study these are presented.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Original recordings or scans from SDS-PAGE gels are provided as source data and deposited with Dryad:This paper does not report original code.

The following data sets were generated
    1. Nicke A
    2. Durner A
    3. Durner E
    (2023) Table 1-source data 1
    Dryad Digital Repository, doi:10.5061/dryad.p8cz8w9tb.

Article and author information

Author details

  1. Anna Durner

    Department of Molecular Biology of Neuronal Signals, LMU Munich, Munich, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Ellis Durner

    Center for Nanoscience, LMU Munich, Munich, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Annette Nicke

    Department of Molecular Biology of Neuronal Signals, LMU Munich, Munich, Germany
    For correspondence
    annette.nicke@lrz.uni-muenchen.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6798-505X

Funding

Deutsche Forschungsgemeinschaft (335447717 - SFB 1328,A15)

  • Annette Nicke

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

Reviewing Editor

  1. Stephan A Pless, University of Copenhagen, Denmark

Version history

  1. Received: August 5, 2022
  2. Preprint posted: September 1, 2022 (view preprint)
  3. Accepted: January 3, 2023
  4. Accepted Manuscript published: January 4, 2023 (version 1)
  5. Version of Record published: January 20, 2023 (version 2)

Copyright

© 2023, Durner 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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  1. Anna Durner
  2. Ellis Durner
  3. Annette Nicke
(2023)
Improved ANAP incorporation and VCF analysis reveals details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain
eLife 12:e82479.
https://doi.org/10.7554/eLife.82479

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https://doi.org/10.7554/eLife.82479

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