Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors

• Published in: Nature communications 2015-08, Vol.6 (1), p.8057, Article 8057
• Main Authors: daCosta, Corrie J. B., Free, Chris R., Sine, Steven M.
• Format: Article
• Language: English
• Subjects: 631/57/2272/2273; 9/74; alpha7 Nicotinic Acetylcholine Receptor - antagonists & inhibitors; alpha7 Nicotinic Acetylcholine Receptor - genetics; alpha7 Nicotinic Acetylcholine Receptor - metabolism; Binding Sites; Bungarotoxins - pharmacology; Gene Expression Regulation - drug effects; HEK293 Cells; Humanities and Social Sciences; Humans; Iodine Radioisotopes; Kinetics; Models, Molecular; multidisciplinary; Mutagenesis, Site-Directed; Mutation; Protein Binding; Protein Conformation; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms9057

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. Since their discovery more than fifty years ago, α-neurotoxins have been used to study acetylcholine receptor-coupled ion channels. Here, daCosta et al . find that toxin binding to a single site of the pentameric α7 receptor blocks function, suggesting the five binding sites are interdependent and the toxin arrests the sites in the inactive conformation.