Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation.

Cécile Pétigny,Mircea Iftinca,Élie Besserer-Offroy,Richard Leduc,Mannix Auger-Messier,Audrey Collette,Brian J Holleran,Hugo Giguère,Christophe Altier,Audrey-Ann Dumont

doi:10.3390/ijms23052502

Abstract

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca2+ homeostasis, and whose functions are modulated by G protein-coupled receptors (GPCR). In this study, we developed bioluminescent resonance energy transfer (BRET) biosensors to better study channel conformational changes following receptor activation. For this study, two intramolecular biosensors, GFP10-TRPC7-RLucII and RLucII-TRPC7-GFP10, were constructed and were assessed following the activation of various GPCRs. We first transiently expressed receptors and the biosensors in HEK293 cells, and BRET levels were measured following agonist stimulation of GPCRs. The activation of GPCRs that engage Gαq led to a Gαq-dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (AT1R), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous AT1R and TRPC7. We detected similar BRET responses in these cells, thus validating the use of the biosensor in physiological conditions. Taken together, our results suggest that activation of Gαq-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.

Highlights

Transient Receptor Potential (TRP) is a superfamily of transmembrane proteins that function as ion channels
We developed intramolecular TRPC7 bioluminescent resonance energy transfer (BRET)-based biosensors to detect TRPC7 conformational changes with Renilla luciferase (RLucII), and a modified version of the green fluorescent protein (GFP10) fused to the N-terminus and the C-terminus, respectively (RLucII-TRPC7-GFP10), or vice versa (GFP10-TRPC7-RlucII) (Figure 1)
As Liu et al developed an intramolecular BRET to monitor TRPC3 activity, we used the same design for TRPC7 [18]

Summary

Introduction

Transient Receptor Potential (TRP) is a superfamily of transmembrane proteins that function as ion channels. They present several motifs, reported to be important for their trafficking and interactions with other proteins or TRPs, such as ankyrin repeat, coil-coiled domains, PSD-95, Discs-large, ZO-1 (PDZ), and calmodulin/IP3 receptor binding (CIRB) domains [2]. TRPC subunits assemble in an oligomeric structure thereby forming homo-tetrameric or hetero-tetrameric non-selective cation channels that play an important role in ion homeostasis. While TRPC channels are reported to be non-selective, they enable Ca2+ entry [3]. TRPC proteins can be divided in two groups, depending on their sequence and functional homologies: (i) TRPC1/4/5 are diacylglycerol (DAG)-insensitive and activated by complex pathways [4], and (ii) TRPC3/6/7 are activated by DAG and its analogs [4,5]

Methods

Results

Conclusion