RESUMO
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.
