TY - JOUR TI - Mutational analysis to explore long-range allosteric couplings involved in a pentameric channel receptor pre-activation and activation AU - Lefebvre, Solène N AU - Taly, Antoine AU - Menny, Anaïs AU - Medjebeur, Karima AU - Corringer, Pierre-Jean A2 - Czajkowski, Cynthia M A2 - Boudker, Olga A2 - Plested, Andrew JR A2 - Brannigan, Grace VL - 10 PY - 2021 DA - 2021/09/30 SP - e60682 C1 - eLife 2021;10:e60682 DO - 10.7554/eLife.60682 UR - https://doi.org/10.7554/eLife.60682 AB - Pentameric ligand-gated ion channels (pLGICs) mediate chemical signaling through a succession of allosteric transitions that are yet not completely understood as intermediate states remain poorly characterized by structural approaches. In a previous study on the prototypic bacterial proton-gated channel GLIC, we generated several fluorescent sensors of the protein conformation that report a fast transition to a pre-active state, which precedes the slower process of activation with pore opening. Here, we explored the phenotype of a series of allosteric mutations, using simultaneous steady-state fluorescence and electrophysiological measurements over a broad pH range. Our data, fitted to a three-state Monod-Wyman-Changeux model, show that mutations at the subunit interface in the extracellular domain (ECD) principally alter pre-activation, while mutations in the lower ECD and in the transmembrane domain principally alter activation. We also show that propofol alters both transitions. Data are discussed in the framework of transition pathways generated by normal mode analysis (iModFit). It further supports that pre-activation involves major quaternary compaction of the ECD, and suggests that activation involves principally a reorganization of a ‘central gating region’ involving a contraction of the ECD β-sandwich and the tilt of the channel lining M2 helix. KW - fluorescence quenching KW - pentameric ligand-gated ion channels KW - intermediate conformation KW - allosteric coupling JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -