A FRET sensor of C-terminal movement reveals VRAC activation by plasma membrane DAG signaling rather than ionic strength

  1. Benjamin König
  2. Yuchen Hao
  3. Sophia Schwartz
  4. Andrew J R Plested
  5. Tobias Stauber  Is a corresponding author
  1. Freie Universität Berlin, Germany
  2. Humboldt Universität zu Berlin, Germany

Abstract

Volume-regulated anion channels (VRACs) are central to cell volume regulation. Recently identified as hetero-hexamers formed by LRRC8 proteins, their activation mechanism remains elusive. Here we measured Förster resonance energy transfer (FRET) between fluorescent proteins fused to the C-termini of LRRC8 subunits. Inter-subunit FRET from LRRC8 complexes tracked VRAC activation. With patch-clamp fluorometry, we confirmed that the cytoplasmic domains rearrange during VRAC opening. With these FRET reporters, we determined VRAC activation, non-invasively, in live cells and their subcompartments. Reduced intracellular ionic strength did not directly activate VRACs, and VRACs were not activated on endomembranes. Instead, pharmacological manipulation of diacylglycerol (DAG), and protein kinase D (PKD) activity, activated or inhibited plasma membrane-localized VRACs. Finally, we resolved previous contradictory reports concerning VRAC activation, using FRET to detect robust activation by PMA that was absent during whole-cell patch clamp. Overall, non-invasive VRAC measurement by FRET is an essential tool for unravelling its activation mechanism.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Benjamin König

    Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Yuchen Hao

    Institute of Biology, Humboldt Universität zu Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Sophia Schwartz

    Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Andrew J R Plested

    Institute of Biology, Humboldt Universität zu Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6062-0832
  5. Tobias Stauber

    Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
    For correspondence
    tobias.stauber@fu-berlin.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0727-6109

Funding

Bundesministerium für Bildung und Forschung (031A314)

  • Tobias Stauber

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

Reviewing Editor

  1. Leon D Islas, Universidad Nacional Autónoma de México, Mexico

Publication history

  1. Received: February 14, 2019
  2. Accepted: June 14, 2019
  3. Accepted Manuscript published: June 18, 2019 (version 1)
  4. Version of Record published: June 27, 2019 (version 2)

Copyright

© 2019, König 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. Benjamin König
  2. Yuchen Hao
  3. Sophia Schwartz
  4. Andrew J R Plested
  5. Tobias Stauber
(2019)
A FRET sensor of C-terminal movement reveals VRAC activation by plasma membrane DAG signaling rather than ionic strength
eLife 8:e45421.
https://doi.org/10.7554/eLife.45421

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