Genetically-encoded BRET probes shed light on ligand bias–induced variable ion selectivity in TRPV1 and P2X5/7

Author:

Chappe Yann Loïck1ORCID,Pierredon Sandra2,Joushomme Alexandre1,Molle Pablo1ORCID,Garenne André1ORCID,Canovi Anne1ORCID,Barbeau Solène3,Poulletier De Gannes Florence1ORCID,Hurtier Annabelle1,Lagroye Isabelle1ORCID,Ducret Thomas3ORCID,Quignard Jean-François3ORCID,Compan Vincent2ORCID,Percherancier Yann1

Affiliation:

1. Laboratory of Integration from Materials to System, CNRS UMR 5218, University of Bordeaux, Talence, F-33400 France

2. Institute of Functional Genomics, CNRS, INSERM UMR 5203, University of Montpellier, Montpellier, F-34094 France

3. Centre de Recherche Cardio-Thoracique de Bordeaux, INSERM U1045, University of Bordeaux, Pessac, F-33600 France

Abstract

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca 2+ and K + concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca 2+ influx than the CAPS-induced K + efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K + efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

Publisher

Proceedings of the National Academy of Sciences

Subject

Multidisciplinary

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