Identification of a pre-active conformation of a pentameric channel receptor

Elife. 2017 Mar 15;6:e23955. doi: 10.7554/eLife.23955.

Abstract

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical signaling through global allosteric transitions. Despite the existence of several high-resolution structures of pLGICs, their dynamical properties remain elusive. Using the proton-gated channel GLIC, we engineered multiple fluorescent reporters, each incorporating a bimane and a tryptophan/tyrosine, whose close distance causes fluorescence quenching. We show that proton application causes a global compaction of the extracellular subunit interface, coupled to an outward motion of the M2-M3 loop near the channel gate. These movements are highly similar in lipid vesicles and detergent micelles. These reorganizations are essentially completed within 2 ms and occur without channel opening at low proton concentration, indicating that they report a pre-active intermediate state in the transition pathway toward activation. This provides a template to investigate the gating of eukaryotic neurotransmitter receptors, for which intermediate states also participate in activation.

Keywords: allostery; biophysics; fluorescence; ligand-gated ion channels; neuroscience; none; structural biology.

MeSH terms

  • Animals
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Bridged Bicyclo Compounds, Heterocyclic / chemistry*
  • Cloning, Molecular
  • Cyanobacteria / chemistry*
  • Cyanobacteria / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Fluorescent Dyes / chemistry
  • Gene Expression
  • Hydrogen-Ion Concentration
  • Kinetics
  • Ligand-Gated Ion Channels / chemistry*
  • Ligand-Gated Ion Channels / genetics
  • Ligand-Gated Ion Channels / metabolism
  • Membrane Potentials / physiology
  • Models, Molecular
  • Mutation
  • Oocytes / cytology
  • Oocytes / metabolism
  • Patch-Clamp Techniques
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Protein Multimerization
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Staining and Labeling / methods
  • Xenopus laevis

Substances

  • Bacterial Proteins
  • Bridged Bicyclo Compounds, Heterocyclic
  • Fluorescent Dyes
  • Ligand-Gated Ion Channels
  • Recombinant Proteins
  • bimanes