Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels

详细信息    查看全文

• 作者：Hermanus Johannes Ruigrok¹ ; ² ; Guillaume Shahid¹ ; ² ; Bertrand Goudeau² ; ³ ; Florence Poulletier de Gannes¹ ; ² ; Emmanuelle Poque-Haro¹ ; ² ; Annabelle Hurtier¹ ; ² ; Isabelle Lagroye¹ ; ² ; ⁴ ; Pierre Vacher² ; ⁵ ; Sté ; phane Arbault² ; ³ ; Neso Sojic² ; ³ ; Bernard Veyret¹ ; ² ; Yann Percherancier¹ ; ² ; ^{yann.percherancier@ims-bordeaux.fr}
• 刊名：Biophysical Journal
• 出版年：2017
• 出版时间：10 January 2017
• 年：2017
• 卷：112
• 期：1
• 页码：87-98
• 全文大小：1430 K
• 卷排序：112

文摘

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 Ca2+-bound CaM but not Ca2+-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.