A FRET sensor of C-terminal movement reveals VRAC activation by plasma membrane DAG signaling rather than ionic strength.
| Autor: | König B; Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany., Hao Y; Institute of Biology, Humboldt Universität zu Berlin, Berlin, Germany.; Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.; NeuroCure, Charité Universitätsmedizin, Berlin, Germany., Schwartz S; Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany., Plested AJ; Institute of Biology, Humboldt Universität zu Berlin, Berlin, Germany.; Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.; NeuroCure, Charité Universitätsmedizin, Berlin, Germany., Stauber T; Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany. |
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| Jazyk: | angličtina |
| Zdroj: | ELife [Elife] 2019 Jun 18; Vol. 8. Date of Electronic Publication: 2019 Jun 18. |
| DOI: | 10.7554/eLife.45421 |
| Abstrakt: | 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 unraveling its activation mechanism. Competing Interests: BK, YH, SS, AP, TS No competing interests declared (© 2019, König et al.) |
| Databáze: | MEDLINE |
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