Title: Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes As Revealed by Intrinsic Tryptophan Fluorescence Quenching
Authors: Billen, Bert ×
Brams, Marijke
Debaveye, Sarah
Remeeva, Alina
Aguiar Alpizar, Yeranddy
Waelkens, Etienne
Kreir, Mohamed
Brüggemann, Andrea
Talavera PĂ©rez, Karel
Nilius, Bernd
Voets, Thomas
Ulens, Chris #
Issue Date: May-2015
Publisher: American Society for Biochemistry and Molecular Biology
Series Title: Journal of Biological Chemistry vol:290 issue:20 pages:12964-74
Article number: jbc.M114.628925
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. While 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 3-D structures are indispensible, as 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 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 green fluorescent protein (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.
ISSN: 0021-9258
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Laboratory of Structural Neurobiology
Laboratory of Ion Channel Research
Laboratory of Protein Phosphorylation and Proteomics
Department of Cellular and Molecular Medicine - miscellaneous
× corresponding author
# (joint) last author

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