title: Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels.
abstract: 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.
authors.affiliation: Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France; Université de Bordeaux, Talence, France.
authors.forename: Hermanus Johannes
authors.initials: HJ
authors.lastname: Ruigrok
authors.affiliation: Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France; Université de Bordeaux, Talence, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Shahid
authors.affiliation: Université de Bordeaux, Talence, France; Institut des Sciences Moleculaires, Centre National de la Recherche Scientifique (CNRS) UMR 5255, NSYSA Group, ENSCBP, Pessac, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Goudeau
authors.affiliation: Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France; Université de Bordeaux, Talence, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Poulletier de Gannes
authors.affiliation: Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France; Université de Bordeaux, Talence, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Poque-Haro
authors.affiliation: Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France; Université de Bordeaux, Talence, France.
authors.forename: Annabelle
authors.initials: A
authors.lastname: Hurtier
authors.affiliation: Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France; Université de Bordeaux, Talence, France; Paris Sciences et Lettres Research University, Paris, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Lagroye
authors.affiliation: Université de Bordeaux, Talence, France; Institut National de la Santé et de la Recherche Médicale (INSERM) U1218, Institut Bergonié, Bordeaux, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Vacher
authors.affiliation: Université de Bordeaux, Talence, France; Institut des Sciences Moleculaires, Centre National de la Recherche Scientifique (CNRS) UMR 5255, NSYSA Group, ENSCBP, Pessac, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Arbault
authors.affiliation: Université de Bordeaux, Talence, France; Institut des Sciences Moleculaires, Centre National de la Recherche Scientifique (CNRS) UMR 5255, NSYSA Group, ENSCBP, Pessac, France.
authors.forename: Neso
authors.initials: N
authors.lastname: Sojic
authors.affiliation: Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France; Université de Bordeaux, Talence, France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Veyret
authors.affiliation: Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France; Université de Bordeaux, Talence, France. Electronic address: yann.percherancier@ims-bordeaux.fr.
authors.forename: Yann
authors.initials: Y
authors.lastname: Percherancier
mesh_terms: D015374:Biosensing Techniques; D002147:Calmodulin / Q000378:metabolism; D004735:Energy Transfer; D057809:HEK293 Cells; D006358:Hot Temperature; D006801:Humans; D008163:Luminescent Measurements; D050916:TRPV Cation Channels / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D050916:TRPV Cation Channels