Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels
| Autor: | Bertrand Goudeau, Stéphane Arbault, Annabelle Hurtier, Guillaume Shahid, Hermanus Ruigrok, Bernard Veyret, Florence Poulletier de Gannes, Pierre Vacher, Isabelle Lagroye, Yann Percherancier, Neso Sojic, Emmanuelle Poque-Haro |
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| Přispěvatelé: | Institut des Sciences Moléculaires (ISM), Université Montesquieu - Bordeaux 4-Université Sciences et Technologies - Bordeaux 1-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de l'intégration, du matériau au système (IMS), Université Sciences et Technologies - Bordeaux 1-Institut Polytechnique de Bordeaux-Centre National de la Recherche Scientifique (CNRS), EPHE PSL Research University (EPHE PSL), Centre National de la Recherche Scientifique (CNRS)-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Université Sciences et Technologies - Bordeaux 1-Université Montesquieu - Bordeaux 4-Institut de Chimie du CNRS (INC), Université Paris sciences et lettres (PSL), Institut Bergonié [Bordeaux], UNICANCER |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
TRPV4 TRPV3 Hot Temperature Calmodulin [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph] Biophysics TRPV1 TRPV Cation Channels Biosensing Techniques TRPV Biological pathway 03 medical and health sciences Transient receptor potential channel 0302 clinical medicine [CHIM.ANAL]Chemical Sciences/Analytical chemistry Humans [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Channels and Transporters ComputingMilieux_MISCELLANEOUS biology [PHYS.PHYS]Physics [physics]/Physics [physics] Chemistry HEK 293 cells [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry Molecular Biology/Biophysics [SPI.ELEC]Engineering Sciences [physics]/Electromagnetism HEK293 Cells 030104 developmental biology Energy Transfer Biochemistry [SDV.TOX]Life Sciences [q-bio]/Toxicology Luminescent Measurements biology.protein 030217 neurology & neurosurgery |
| Zdroj: | Biophysical Journal Biophysical Journal, Biophysical Society, 2017, 112 (1), pp.87. ⟨10.1016/j.bpj.2016.11.3197⟩ Biophysical Journal, Biophysical Society, 2017, 112 (1), pp.87-98. ⟨10.1016/j.bpj.2016.11.3197⟩ |
| ISSN: | 0006-3495 1542-0086 |
| DOI: | 10.1016/j.bpj.2016.11.3197⟩ |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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