Mapping two neurosteroid-modulatory sites in the prototypic pentameric ligand-gated ion channel GLIC

J Biol Chem. 2018 Feb 23;293(8):3013-3027. doi: 10.1074/jbc.RA117.000359. Epub 2018 Jan 4.


Neurosteroids are endogenous sterols that potentiate or inhibit pentameric ligand-gated ion channels (pLGICs) and can be effective anesthetics, analgesics, or anti-epileptic drugs. The complex effects of neurosteroids on pLGICs suggest the presence of multiple binding sites in these receptors. Here, using a series of novel neurosteroid-photolabeling reagents combined with top-down and middle-down mass spectrometry, we have determined the stoichiometry, sites, and orientation of binding for 3α,5α-pregnane neurosteroids in the Gloeobacter ligand-gated ion channel (GLIC), a prototypic pLGIC. The neurosteroid-based reagents photolabeled two sites per GLIC subunit, both within the transmembrane domain; one site was an intrasubunit site, and the other was located in the interface between subunits. By using reagents with photoreactive groups positioned throughout the neurosteroid backbone, we precisely map the orientation of neurosteroid binding within each site. Amino acid substitutions introduced at either site altered neurosteroid modulation of GLIC channel activity, demonstrating the functional role of both sites. These results provide a detailed molecular model of multisite neurosteroid modulation of GLIC, which may be applicable to other mammalian pLGICs.

Keywords: allosteric regulation; ion channel; mass spectrometry (MS); molecular pharmacology; neurosteroid; photoaffinity labeling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Cyanobacteria
  • Desoxycorticosterone / analogs & derivatives*
  • Desoxycorticosterone / chemistry
  • Desoxycorticosterone / metabolism
  • Hydroxylation
  • Kinetics
  • Ligand-Gated Ion Channels / chemistry
  • Ligand-Gated Ion Channels / genetics
  • Ligand-Gated Ion Channels / metabolism*
  • Ligands
  • Models, Molecular*
  • Molecular Conformation
  • Molecular Docking Simulation
  • Mutagenesis, Site-Directed
  • Neurotransmitter Agents / chemistry
  • Neurotransmitter Agents / metabolism*
  • Photoaffinity Labels / chemistry
  • Point Mutation
  • Pregnanes / chemistry
  • Pregnanes / metabolism*
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping
  • Protein Multimerization
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism


  • Bacterial Proteins
  • Ligand-Gated Ion Channels
  • Ligands
  • Neurotransmitter Agents
  • Photoaffinity Labels
  • Pregnanes
  • Recombinant Proteins
  • Desoxycorticosterone
  • tetrahydrodeoxycorticosterone

Associated data

  • PDB/4HFI
  • PDB/4FHI