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

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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.

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

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Benjamin König

Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany

Competing interests
The authors declare that no competing interests exist.
Yuchen Hao

Institute of Biology, Humboldt Universität zu Berlin, Berlin, Germany

Competing interests
The authors declare that no competing interests exist.
Sophia Schwartz

Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany

Competing interests
The authors declare that no competing interests exist.
Andrew J R Plested

Institute of Biology, Humboldt Universität zu Berlin, Berlin, Germany

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-6062-0832
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.

"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.

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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.