Dataset Information

Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation.

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 Gα_q led to a Gα_q-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 Gα_q-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.

SUBMITTER: Petigny C

PROVIDER: S-EPMC8910688 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation.

Pétigny Cécile C Dumont Audrey-Ann AA Giguère Hugo H Collette Audrey A Holleran Brian J BJ Iftinca Mircea M Altier Christophe C Besserer-Offroy Élie É Auger-Messier Mannix M Leduc Richard R

International journal of molecular sciences 20220224 5

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca<sup>2+</sup> 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 followin ...[more]

PMID: 35269644

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Project description:We present a hybrid computational methodology to predict multiple energetically accessible conformations for G protein-coupled receptors (GPCRs) that might play a role in binding to ligands and different signaling partners. To our knowledge, this method, termed ActiveGEnSeMBLE, enables the first quantitative energy profile for GPCR activation that is consistent with the qualitative profile deduced from experiments. ActiveGEnSeMBLE starts with a systematic coarse grid sampling of helix tilts/rotations (∼13 trillion transmembrane-domain conformations) and selects the conformational landscape based on energy. This profile identifies multiple potential active-state energy wells, with the TM3-TM6 intracellular distance as an approximate activation coordinate. These energy wells are then sampled locally using a finer grid to find locally minimized conformation in each energy well. We validate this strategy using the inactive and active experimental structures of β2 adrenergic receptor (hβ2AR) and M2 muscarinic acetylcholine receptor. Structures of membrane-embedded hβ2AR along its activation coordinate are subjected to molecular-dynamics simulations for relaxation and interaction energy analysis to generate a quantitative energy landscape for hβ2AR activation. This landscape reveals several metastable states along this coordinate, indicating that for hβ2AR, the agonist alone is not enough to stabilize the active state and that the G protein is necessary, consistent with experimental observations. The method's application to somatostatin receptor SSTR5 (no experimental structure available) shows that to predict an active conformation it is better to start from an inactive structure template based on a close homolog than to start from an active template based on a distant homolog. The energy landscape for hSSTR5 activation is consistent with hβ2AR in the role of the G protein. These results demonstrate the utility of the ActiveGEnSeMBLE method for predicting multiple conformations along the pathways for activating GPCRs and the corresponding energy landscapes, thereby providing detailed structural insights into the initial molecular events of GPCR function that are not easily accessible by experiments.

Dataset Information

Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation.

Publications

Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation.

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