Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors

Nat Commun. 2015 Aug 18;6:8057. doi: 10.1038/ncomms9057.

Abstract

α-Bungarotoxin (α-Btx) binds to the five agonist binding sites on the homopentameric α7-acetylcholine receptor, yet the number of bound α-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for α-Btx blockade, we generate receptors comprised of wild-type and α-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with α-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by α-Btx and accessible to the agonist. Given structural evidence that α-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five α7 subunits are interdependent and maintain conformational symmetry in the open channel state.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Binding Sites
  • Bungarotoxins / pharmacology*
  • Gene Expression Regulation / drug effects
  • HEK293 Cells
  • Humans
  • Iodine Radioisotopes
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Binding
  • Protein Conformation
  • alpha7 Nicotinic Acetylcholine Receptor / antagonists & inhibitors*
  • alpha7 Nicotinic Acetylcholine Receptor / genetics
  • alpha7 Nicotinic Acetylcholine Receptor / metabolism

Substances

  • Bungarotoxins
  • Iodine Radioisotopes
  • alpha7 Nicotinic Acetylcholine Receptor