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  • Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching

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    Author(s)
    Billen, Bert
    Brams, Marijke
    Debaveye, Sarah
    Remeeva, Alina
    Alpizar, Yeranddy A
    Waelkens, Etienne
    Kreir, Mohamed
    Brueggemann, Andrea
    Talavera, Karel
    Nilius, Bernd
    Voets, Thomas
    Ulens, Chris
    Griffith University Author(s)
    Nilius, Bernd
    Year published
    2015
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    Abstract
    TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures ...
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    TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.
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    Journal Title
    Journal of Biological Chemistry
    Volume
    290
    Issue
    20
    DOI
    https://doi.org/10.1074/jbc.M114.628925
    Copyright Statement
    © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported (CC BY 3.0) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
    Subject
    Chemical sciences
    Biological sciences
    Biomedical and clinical sciences
    Science & Technology
    Life Sciences & Biomedicine
    Biochemistry & Molecular Biology
    ANKYRIN REPEAT DOMAIN
    SIZE-EXCLUSION CHROMATOGRAPHY
    Publication URI
    http://hdl.handle.net/10072/391440
    Collection
    • Journal articles

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