Ligand Binding at the 4-4 Agonist-Binding Site of the 42 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State

• Published in: PloS one Vol. 11; no. 8; p. e0161154
• Main Authors: Indurthi, Dinesh C, Lewis, Trevor M, Ahring, Philip K, Balle, Thomas, Chebib, Mary, Absalom, Nathan L
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 23.08.2016; Public Library of Science (PLoS)
• Subjects: Acetylcholine - metabolism; Acetylcholine - pharmacology; Acetylcholine receptors; Animals; Azetidines - pharmacology; Binding Sites; Brain research; Cell Membrane - drug effects; Cell Membrane - genetics; Humans; Ion Channel Gating - genetics; Ligand binding (Biochemistry); Ligands; Molecular Conformation - drug effects; Oocytes - drug effects; Oocytes - growth & development; Oocytes - metabolism; Oxadiazoles - pharmacology; Properties; Protein Binding; Protein Isoforms - drug effects; Pyridines - pharmacology; Receptors, Nicotinic - genetics; Receptors, Nicotinic - metabolism; Testing; Xenopus laevis - genetics; Xenopus laevis - growth & development
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0161154

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant subtype in the brain and exists in two functional stoichiometries: (α4)3(β2)2 and (α4)2(β2)3. A distinct feature of the (α4)3(β2)2 receptor is the biphasic activation response to the endogenous agonist acetylcholine, where it is activated with high potency and low efficacy when two α4-β2 binding sites are occupied and with low potency/high efficacy when a third α4-α4 binding site is occupied. Further, exogenous ligands can bind to the third α4-α4 binding site and potentiate the activation of the receptor by ACh that is bound at the two α4-β2 sites. We propose that perturbations of the recently described pre-activation step when a third binding site is occupied are a key driver of these distinct activation properties. To investigate this, we used a combination of simple linear kinetic models and voltage clamp electrophysiology to determine whether transitions into the pre-activated state were increased when three binding sites were occupied. We separated the binding at the two different sites with ligands selective for the α4-β2 site (Sazetidine-A and TC-2559) and the α4-α4 site (NS9283) and identified that when a third binding site was occupied, changes in the concentration-response curves were best explained by an increase in transitions into a pre-activated state. We propose that perturbations of transitions into a pre-activated state are essential to explain the activation properties of the (α4)3(β2)2 receptor by acetylcholine and other ligands. Considering the widespread clinical use of benzodiazepines, this discovery of a conserved mechanism that benzodiazepines and ACh potentiate receptor activation via a third binding site can be exploited to develop therapeutics with similar properties at other cys-loop receptors.