Gating transitions of the glycine receptor using combined electrophysiology, fluorescence and MD simulations – GlyTrans

The glycine receptor (GlyR) is a ligand-gated ion channel mediating neuronal communication and is a promising pharmacological target for treatment of chronic pain, cancer and hyperekplexia. Numerous cryo-EM structures of GlyR with atomic resolution have been published. However, these structures were obtained in non-physiological conditions questioning their physiological significance. Conversely, functional measurements like electrophysiology probe physiologically relevant states but little structural information. To bridge the gap between structure and function, we propose a synergistic combination of electrophysiology, fluorescence, and molecular dynamics (MD) simulations to obtain a comprehensive multi-step model of alpha1-GlyR activation and desensitization: 1/ To explore the conformational dynamics of the protein at the cell membrane, we will employ voltage-clamp fluorometry (VCF). VCF provides simultaneous measurements of channel opening by electrophysiology and local protein motions by fluorescence. By incorporating the unnatural fluorescent amino acid ANAP, we will map the entire protein structure and illuminate the gating motions. 2/ In parallel, we will perform a thorough exploration of the conformational space of the receptor by MD simulations and construct a Markov-state model (MSM) of activation and desensitization. It will illuminate the stable states and the allosteric transition pathway(s) with atomic resolution, including metastable intermediates. 3/ Conformations extracted from MSM will be analyzed in terms of local conformations, ion-channel state and predicted pharmacological profile to assign them to states illuminated by VCF. The MSM/VCF analysis will be repeated with modulatory ligands and/or pathological mutations. This project will decipher the mechanism of action of ligands that may open to original pharmacological strategies, and will characterize in depth the phenotype of pathological mutations related to hyperekplexia and cancer.