Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada.
Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada.
Int J Mol Sci. 2022 Feb 24;23(5):2502. doi: 10.3390/ijms23052502.
Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca 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 Gα led to a Gα-dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (ATR), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous ATR 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 Gα-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.
瞬时受体电位经典型 (TRPC) 通道是参与调节钙稳态的膜蛋白，其功能受 G 蛋白偶联受体 (GPCR) 调节。在这项研究中，我们开发了生物发光共振能量转移 (BRET) 生物传感器，以更好地研究受体激活后通道构象的变化。为此，我们构建了两个分子内生物传感器 GFP10-TRPC7-RLucII 和 RLucII-TRPC7-GFP10，并在各种 GPCR 激活后对其进行了评估。我们首先在 HEK293 细胞中转染受体和生物传感器，然后在 GPCR 激动剂刺激后测量 BRET 水平。与 Gα 结合的 GPCR 的激活导致功能性 TRPC7 生物传感器的 Gα 依赖性 BRET 反应。关注血管紧张素 II 型 1 受体 (ATR)，我们在表达内源性 ATR 和 TRPC7 的大鼠新生心肌成纤维细胞中测试了 GFP10-TRPC7-RLucII。我们在这些细胞中检测到类似的 BRET 反应，从而验证了该生物传感器在生理条件下的使用。总之，我们的结果表明，Gα 偶联受体的激活诱导新型功能性 TRPC7 BRET 生物传感器发生构象变化。
