title: Role of the Cys Loop and Transmembrane Domain in the Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors.
abstract: Allosteric modulators of pentameric ligand-gated ion channels are thought to act on elements of the pathways that couple agonist binding to channel gating. Using α4β2 nicotinic acetylcholine receptors and the α4β2-selective positive modulators 17β-estradiol (βEST) and desformylflustrabromine (dFBr), we have identified pathways that link the binding sites for these modulators to the Cys loop, a region that is critical for channel gating in all pentameric ligand-gated ion channels. Previous studies have shown that the binding site for potentiating βEST is in the C-terminal (post-M4) region of the α4 subunit. Here, using homology modeling in combination with mutagenesis and electrophysiology, we identified the binding site for potentiating dFBr on the top half of a cavity between the third (M3) and fourth transmembrane (M4) α-helices of the α4 subunit. We found that the binding sites for βEST and dFBr communicate with the Cys loop, through interactions between the last residue of post-M4 and Phe170of the conserved FPF sequence of the Cys loop, and that these interactions affect potentiating efficacy. In addition, interactions between a residue in M3 (Tyr309) and Phe167, a residue adjacent to the Cys loop FPF motif, also affect dFBr potentiating efficacy. Thus, the Cys loop acts as a key control element in the allosteric transduction pathway for potentiating βEST and dFBr. Overall, we propose that positive allosteric modulators that bind the M3-M4 cavity or post-M4 region increase the efficacy of channel gating through interactions with the Cys loop.
authors.affiliation: From the Department of Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom.
authors.forename: Constanza
authors.initials: C
authors.lastname: Alcaino
authors.affiliation: the Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Musgaard
authors.affiliation: From the Department of Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom.
authors.forename: Teresa
authors.initials: T
authors.lastname: Minguez
authors.affiliation: From the Department of Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom.
authors.forename: Simone
authors.initials: S
authors.lastname: Mazzaferro
authors.affiliation: the Faculty of Sciences, University of Chile, Santiago 7800003, Chile, and.
authors.forename: Manuel
authors.initials: M
authors.lastname: Faundez
authors.affiliation: the Departamento de Ciencias Quimicas y Recursos Naturales, Facultad de Ingenieria y Ciencias, Universidad de la Frontera, Temuco 4811230, Chile.
authors.forename: Patricio
authors.initials: P
authors.lastname: Iturriaga-Vasquez
authors.affiliation: the 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, Oxford Brookes University, Oxford OX3 0BP, United Kingdom, ibermudez@brookes.ac.uk.
authors.forename: Isabel
authors.identifier: http://orcid.org/0000-0001-7692-1509
authors.initials: I
authors.lastname: Bermudez
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D004958:Estradiol / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D004958:Estradiol
keywords: C-terminal domain (carboxyl tail domain, CTD); Cys-loop receptor; nicotinic acetylcholine receptors (nAChR); signal transduction; transmembrane domain