title: Non-equivalent ligand selectivity of agonist sites in (α4β2)2α4 nicotinic acetylcholine receptors: a key determinant of agonist efficacy.
abstract: The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR type in the brain, and this receptor type exists in alternate (α4β2)2α4 and (α4β2)2β2 forms, which are activated by agonists with strikingly differing efficacies. Recent breakthroughs have identified an additional operational agonist binding site in the (α4β2)2α4 nAChR that is responsible for the signature sensitivity of this receptor to activation by agonists, yet the structural mechanisms determining agonist efficacy at this receptor type are not yet fully understood. In this study, we characterized the ligand selectivity of the individual agonist sites of the (α4β2)2α4 nAChR to determine whether differences in agonist selectivity influence agonist efficacy. Applying the substituted cysteine accessibility method to individual agonist sites in concatenated (α4β2)2α4 receptors, we determined the agonist selectivity of the agonist sites of the (α4β2)2α4 receptor. We show that (a) accessibility of substituted cysteines to covalent modification by methanesulfonate reagent depends on the agonist site at which the modification occurs and (b) that agonists such as sazetidine-A and TC-2559 are excluded from the site at the α4/α4 interface. Given that additional binding to the agonist site in the α4/α4 interface increases acetylcholine efficacy and that agonists excluded from the agonist site at the α4/α4 interface behave as partial agonists, we conclude that the ability to engage all agonist sites in (α4β2)2α4 nAChRs is a key determinant of agonist efficacy. The findings add another level of complexity to the structural mechanisms that govern agonist efficacy in heteromeric nAChRs and related ligand-gated ion channels.
authors.affiliation: From the Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, United Kingdom.
authors.forename: Simone
authors.initials: S
authors.lastname: Mazzaferro
authors.affiliation: From the Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, United Kingdom.
authors.forename: Federica
authors.initials: F
authors.lastname: Gasparri
authors.affiliation: From the Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, United Kingdom.
authors.forename: Karina
authors.initials: K
authors.lastname: New
authors.affiliation: From the Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, United Kingdom.
authors.forename: Constanza
authors.initials: C
authors.lastname: Alcaino
authors.affiliation: the Department of Chemistry, Faculty of Sciences, University of Chile, Santiago, Chile.
authors.forename: Manuel
authors.initials: M
authors.lastname: Faundez
authors.affiliation: the Department of Chemistry, Faculty of Sciences, University of Chile, Santiago, Chile.
authors.forename: Patricio
authors.initials: P
authors.lastname: Iturriaga Vasquez
authors.affiliation: Department of Biology, College of Science, United Arab Emirates University, Al Ain, United Arab Emirates, and.
authors.forename: Ranjit
authors.initials: R
authors.lastname: Vijayan
authors.affiliation: Structural Bioinformatics and Computational Biochemistry, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
authors.forename: Philip C
authors.initials: PC
authors.lastname: Biggin
authors.affiliation: From the Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, United Kingdom, ibermudez@brookes.ac.uk.
authors.forename: Isabel
authors.initials: I
authors.lastname: Bermudez
mesh_terms: D000818:Animals; D008024:Ligands; D018722:Nicotinic Agonists / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D008024:Ligands; D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2
keywords: Cys-loop Receptor; Electrophysiology; Ligand-binding Protein; Nicotinic Acetylcholine Receptor (nAChR); Receptor Structure-Function