Mutational analysis to explore long-range allosteric couplings involved in a pentameric channel receptor pre-activation and activation.

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  • Additional Information
    • Source:
      Publisher: eLife Sciences Publications, Ltd Country of Publication: England NLM ID: 101579614 Publication Model: Electronic Cited Medium: Internet ISSN: 2050-084X (Electronic) Linking ISSN: 2050084X NLM ISO Abbreviation: Elife Subsets: MEDLINE
    • Publication Information:
      Original Publication: Cambridge, UK : eLife Sciences Publications, Ltd., 2012-
    • Subject Terms:
      DNA Mutational Analysis* ; Ion Channel Gating* ; Mutation*; Bacterial Proteins/*metabolism ; Cyanobacteria/*metabolism ; Ligand-Gated Ion Channels/*metabolism; Bacterial Proteins/chemistry ; Bacterial Proteins/genetics ; Cyanobacteria/genetics ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Ligand-Gated Ion Channels/chemistry ; Ligand-Gated Ion Channels/genetics ; Models, Biological ; Molecular Docking Simulation ; Protein Conformation ; Structure-Activity Relationship ; Time Factors
    • Abstract:
      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.
      Competing Interests: SL, AT, AM, KM, PC No competing interests declared
      (© 2021, Lefebvre et al.)
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    • Contributed Indexing:
      Keywords: E. coli; allosteric coupling; fluorescence quenching; intermediate conformation; molecular biophysics; neuroscience; pentameric ligand-gated ion channels; structural biology; xenopus
    • Accession Number:
      0 (Bacterial Proteins)
      0 (Ligand-Gated Ion Channels)
    • Subject Terms:
      Gloeobacter violaceus
    • Publication Date:
      Date Created: 20210930 Date Completed: 20211027 Latest Revision: 20220107
    • Publication Date:
      20221213
    • Accession Number:
      PMC8504973
    • Accession Number:
      10.7554/eLife.60682
    • Accession Number:
      34590583