title: Different ligands of the TRPV3 cation channel cause distinct conformational changes as revealed by intrinsic tryptophan fluorescence quenching.
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 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.
authors.affiliation: From the Laboratory of Structural Neurobiology and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 601, 3000 Leuven, Belgium, bert.billen@med.kuleuven.be.
authors.forename: Bert
authors.initials: B
authors.lastname: Billen
authors.affiliation: From the Laboratory of Structural Neurobiology and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 601, 3000 Leuven, Belgium.
authors.forename: Marijke
authors.initials: M
authors.lastname: Brams
authors.affiliation: From the Laboratory of Structural Neurobiology and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 601, 3000 Leuven, Belgium.
authors.forename: Sarah
authors.initials: S
authors.lastname: Debaveye
authors.affiliation: From the Laboratory of Structural Neurobiology and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 601, 3000 Leuven, Belgium.
authors.forename: Alina
authors.initials: A
authors.lastname: Remeeva
authors.affiliation: the Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 802, 3000 Leuven, Belgium.
authors.forename: Yeranddy A
authors.initials: YA
authors.lastname: Alpizar
authors.affiliation: the Laboratory of Protein Phosphorylation and Proteomics, Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 901, 3000 Leuven, Belgium, and.
authors.forename: Etienne
authors.initials: E
authors.lastname: Waelkens
authors.affiliation: Nanion Technologies GmbH, Gabrielenstrasse 9, D-80636 Munich, Germany.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Kreir
authors.affiliation: Nanion Technologies GmbH, Gabrielenstrasse 9, D-80636 Munich, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Brüggemann
authors.affiliation: the Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 802, 3000 Leuven, Belgium.
authors.forename: Karel
authors.initials: K
authors.lastname: Talavera
authors.affiliation: the Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 802, 3000 Leuven, Belgium.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nilius
authors.affiliation: the Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 802, 3000 Leuven, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Voets
authors.affiliation: From the Laboratory of Structural Neurobiology and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 601, 3000 Leuven, Belgium.
authors.forename: Chris
authors.initials: C
authors.lastname: Ulens
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D050916:TRPV Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D008024:Ligands; D011993:Recombinant Fusion Proteins; D050916:TRPV Cation Channels; C459445:TRPV3 protein, human; D049452:Green Fluorescent Proteins; D014364:Tryptophan
keywords: camphor; fluorescence; human TRPV3; icilin; membrane protein; membrane reconstitution; protein purification; quenching; transient receptor potential channels (TRP channels)
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