Ruigrok Hermanus Johannes, Shahid Guillaume, Goudeau Bertrand, Poulletier de Gannes Florence, Poque-Haro Emmanuelle, Hurtier Annabelle, Lagroye Isabelle, Vacher Pierre, Arbault Stéphane, Sojic Neso, Veyret Bernard, Percherancier Yann
Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France; Université de Bordeaux, Talence, France.
Université de Bordeaux, Talence, France; Institut des Sciences Moleculaires, Centre National de la Recherche Scientifique (CNRS) UMR 5255, NSYSA Group, ENSCBP, Pessac, France.
Biophys J. 2017 Jan 10;112(1):87-98. doi: 10.1016/j.bpj.2016.11.3197.
Multiplexed bioluminescence resonance energy transfer (BRET) assays were developed to monitor the activation of several functional transient receptor potential (TRP) channels in live cells and in real time. We probed both TRPV1 intramolecular rearrangements and its interaction with Calmodulin (CaM) under activation by chemical agonists and temperature. Our BRET study also confirmed that: (1) capsaicin and heat promoted distinct transitions, independently coupled to channel gating, and that (2) TRPV1 and Ca-bound CaM but not Ca-free CaM were preassociated in resting live cells, while capsaicin activation induced both the formation of more TRPV1/CaM complexes and conformational changes. The BRET assay, based on the interaction with Calmodulin, was successfully extended to TRPV3 and TRPV4 channels. We therefore developed a full-spectral three-color BRET assay for analyzing the specific activation of each of the three TRPV channels in a single sample. Such key improvement in BRET measurement paves the way for the simultaneous monitoring of independent biological pathways in live cells.
开发了多重生物发光共振能量转移（BRET）分析方法，以实时监测活细胞中几种功能性瞬时受体电位（TRP）通道的激活情况。我们探究了化学激动剂和温度激活下TRPV1的分子内重排及其与钙调蛋白（CaM）的相互作用。我们的BRET研究还证实：（1）辣椒素和热促进了不同的转变，这些转变独立地与通道门控相关联；（2）在静息活细胞中，TRPV1与结合钙的CaM而非未结合钙的CaM预先结合，而辣椒素激活既诱导了更多TRPV1/CaM复合物的形成，也引起了构象变化。基于与钙调蛋白相互作用的BRET分析方法成功扩展到了TRPV3和TRPV4通道。因此，我们开发了一种全光谱三色BRET分析方法，用于分析单个样品中三种TRP通道各自的特异性激活情况。BRET测量中的这一关键改进为同时监测活细胞中的独立生物学途径铺平了道路。
