Billen Bert, Brams Marijke, Debaveye Sarah, Remeeva Alina, Alpizar Yeranddy A, Waelkens Etienne, Kreir Mohamed, Brüggemann Andrea, Talavera Karel, Nilius Bernd, Voets Thomas, Ulens Chris
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,
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.
J Biol Chem. 2015 May 15;290(20):12964-74. doi: 10.1074/jbc.M114.628925. Epub 2015 Mar 31.
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.
瞬时受体电位香草酸亚型3（TRPV3）是一种热敏离子通道，主要表达于皮肤、鼻子和舌头的上皮组织中。该通道与环境温度感知、炎症组织中的痛觉过敏、皮肤致敏和毛发生长有关。尽管瞬时受体电位（TRP）通道的研究极大地增进了我们对伤害感受和温度感知生理机制的理解，但这些离子通道的分子机制仍 largely难以捉摸。为了更好地理解TRP通道的功能特性和作用机制，高分辨率三维结构是不可或缺的，因为它们将在原子水平上提供对结构细节的必要见解。然而，膜蛋白的结构研究目前受到蛋白质纯化和建立合适结晶条件方面困难的阻碍。在本报告中，我们提出了一种利用C端绿色荧光蛋白（GFP）融合来纯化膜蛋白的新方案。使用该方案，我们纯化了人TRPV3。我们通过对重组通道进行平面膜片钳记录和固有色氨酸荧光光谱法表明，纯化后的蛋白是一种具有与天然通道相似特性的全功能离子通道。通过固有色氨酸荧光光谱法，我们揭示了不同配体与该通道分子相互作用中的明显差异。总之，本研究提供了强大的工具来拓宽我们对配体与TRPV通道相互作用的理解，纯化的人TRPV3的可得性为进一步的结构和功能研究开辟了前景。
