title: Ligand Binding at the 4-4 Agonist-Binding Site of the 42 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State.
abstract: 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.
authors.affiliation: Faculty of Pharmacy, University of Sydney, NSW, 2006, Australia.
authors.forename: Dinesh C
authors.initials: DC
authors.lastname: Indurthi
authors.affiliation: School of Medical Sciences, University of NSW, Kensington, NSW, 2052, Australia.
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Lewis
authors.affiliation: Faculty of Pharmacy, University of Sydney, NSW, 2006, Australia.
authors.forename: Philip K
authors.initials: PK
authors.lastname: Ahring
authors.affiliation: Faculty of Pharmacy, University of Sydney, NSW, 2006, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Balle
authors.affiliation: Faculty of Pharmacy, University of Sydney, NSW, 2006, Australia.
authors.forename: Mary
authors.initials: M
authors.lastname: Chebib
authors.affiliation: Faculty of Pharmacy, University of Sydney, NSW, 2006, Australia.
authors.forename: Nathan L
authors.initials: NL
authors.lastname: Absalom
mesh_terms: D000109:Acetylcholine / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001384:Azetidines / Q000494:pharmacology; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics*; D008024:Ligands; D008968:Molecular Conformation / Q000187:drug effects; D009865:Oocytes / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D010069:Oxadiazoles / Q000494:pharmacology; D011485:Protein Binding; D020033:Protein Isoforms / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics / Q000254:growth & development
chemical_list: C561896:3-(3-(pyridine-3-yl)-1,2,4-oxadiazol-5-yl)benzonitrile; D001384:Azetidines; D008024:Ligands; D010069:Oxadiazoles; D020033:Protein Isoforms; D011725:Pyridines; D011978:Receptors, Nicotinic; C419442:TC 2559; C514816:sazetidine-A; D000109:Acetylcholine