Expression and Purification of the Pain Receptor TRPV1 for Spectroscopic Analysis.
Expression and Purification of the Pain Receptor TRPV1 for Spectroscopic Analysis.
复制标题
用于光谱分析的疼痛受体 TRPV1 的表达和纯化。
DOI：
10.1038/s41598-017-10426-7
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发表时间：
2017
影响因子：
4.6
通讯作者：
Cordero-Morales,JulioF
中科院分区：
文献类型：
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作者：
Velisetty,Phanindra;Stein,RichardA;Sierra-Valdez,FranciscoJ;Vásquez,Valeria;Cordero-Morales,JulioF
瞬时受体电位香草素1（TRPV 1）通道是有害刺激引起疼痛的细胞机制的重要组成部分。TRPV 1的功能和结构特征揭示了香草素的激活，但温度和质子门控的机制在很大程度上仍然未知。需要光谱方法来理解TRPV 1将不同刺激转化为通道开放的机制。在这里，我们已经设计了一个最小的半胱氨酸少大鼠TRPV 1结构（eTRPV 1），可以稳定纯化和重建光谱研究。TRPV 1结构的生物物理分析表明，S5孔螺旋环影响蛋白质的稳定性和香草素和质子的反应，但不热敏感性。半胱氨酸突变体保留了双电子-电子共振（DEER）和电子顺磁共振（EPR）光谱的功能和稳定性。在闭合状态下的DEER测量表明，eTRPV 1报告细胞外前庭中的距离，相当于在apo TRPV 1结构中观察到的距离。EPR测量显示出一个独特的模式的流动性和光谱特征，在洗涤剂和脂质体，在孔域的残基，同意他们的位置在TRPV 1结构。我们的研究结果为TRPV 1的系统表征奠定了基础，该研究使用光谱方法揭示了与热依赖性和配体依赖性门控相容的构象变化。
The transient receptor potential vanilloid 1 (TRPV1) channel is an essential component of the cellular mechanism through which noxious stimuli evoke pain. Functional and structural characterizations of TRPV1 shed light on vanilloid activation, yet the mechanisms for temperature and proton gating remain largely unknown. Spectroscopic approaches are needed to understand the mechanisms by which TRPV1 translates diverse stimuli into channel opening. Here, we have engineered a minimal cysteine-less rat TRPV1 construct (eTRPV1) that can be stably purified and reconstituted for spectroscopic studies. Biophysical analyses of TRPV1 constructs reveal that the S5-pore helix loop influences protein stability and vanilloid and proton responses, but not thermal sensitivity. Cysteine mutants retain function and stability for double electron-electron resonance (DEER) and electron paramagnetic resonance (EPR) spectroscopies. DEER measurements in the closed state demonstrate that eTRPV1 reports distances in the extracellular vestibule, equivalent to those observed in the apo TRPV1 structure. EPR measurements show a distinct pattern of mobilities and spectral features, in detergent and liposomes, for residues at the pore domain that agree with their location in the TRPV1 structure. Our results set the stage for a systematic characterization of TRPV1 using spectroscopic approaches to reveal conformational changes compatible with thermal- and ligand-dependent gating.