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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 unraveling its activation mechanism.

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      Most cited references 62

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      Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
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        μManager is an open-source, cross-platform desktop application, to control a wide variety of motorized microscopes, scientific cameras, stages, illuminators, and other microscope accessories. Since its inception in 2005, μManager has grown to support a wide range of microscopy hardware and is now used by thousands of researchers around the world. The application provides a mature graphical user interface and offers open programming interfaces to facilitate plugins and scripts. Here, we present a guide to using some of the recently added advanced μManager features, including hardware synchronization, simultaneous use of multiple cameras, projection of patterned light onto a specimen, live slide mapping, imaging with multi-well plates, particle localization and tracking, and high-speed imaging.
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          Physiology of cell volume regulation in vertebrates.

          The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits a wide array of signaling events, leading to protective (e.g., cytoskeletal rearrangement) and adaptive (e.g., altered expression of osmolyte transporters and heat shock proteins) measures and, in most cases, activation of volume regulatory osmolyte transport. After acute swelling, cell volume is regulated by the process of regulatory volume decrease (RVD), which involves the activation of KCl cotransport and of channels mediating K(+), Cl(-), and taurine efflux. Conversely, after acute shrinkage, cell volume is regulated by the process of regulatory volume increase (RVI), which is mediated primarily by Na(+)/H(+) exchange, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) channels. Here, we review in detail the current knowledge regarding the molecular identity of these transport pathways and their regulation by, e.g., membrane deformation, ionic strength, Ca(2+), protein kinases and phosphatases, cytoskeletal elements, GTP binding proteins, lipid mediators, and reactive oxygen species, upon changes in cell volume. We also discuss the nature of the upstream elements in volume sensing in vertebrate organisms. Importantly, cell volume impacts on a wide array of physiological processes, including transepithelial transport; cell migration, proliferation, and death; and changes in cell volume function as specific signals regulating these processes. A discussion of this issue concludes the review.
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            Author and article information

            Affiliations
            [1 ]deptInstitute of Chemistry and Biochemistry Freie Universität Berlin BerlinGermany
            [2 ]deptInstitute of Biology Humboldt Universität zu Berlin BerlinGermany
            [3 ]Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP) BerlinGermany
            [4 ]deptNeuroCure Charité Universitätsmedizin BerlinGermany
            Universidad Nacional Autónoma de México Mexico
            The University of Texas at Austin United States
            Universidad Nacional Autónoma de México Mexico
            Universidad Nacional Autónoma de México Mexico
            Contributors
            ORCID: https://orcid.org/0000-0003-0727-6109
            Role: Reviewing Editor,
            Universidad Nacional Autónoma de México Mexico
            Role: Senior Editor,
            The University of Texas at Austin United States
            Journal
            eLife
            Elife
            eLife
            eLife
            eLife Sciences Publications, Ltd
            2050-084X
            18 June 2019
            2019
            : 8
            31210638 6597245 45421 10.7554/eLife.45421
            © 2019, König et al

            This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

            Product
            Funding
            Funded by: FundRef http://dx.doi.org/10.13039/501100002347, Bundesministerium für Bildung und Forschung;
            Award ID: 031A314
            Award Recipient :
            The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
            Categories
            Research Article
            Cell Biology
            Structural Biology and Molecular Biophysics
            Custom metadata
            VRAC activation, observed with a FRET sensor of intracellular LRRC8-domains movement during gating and by fluorometry, requires plasma membrane localization and diacylglycerol signaling, but is independent of intracellular ionic strength.

            Life sciences

            volume regulation, ionic strength, fret, lrrc8 heteromer, vsor, human, channel gating

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