title: Probing the structure of the uncoupled nicotinic acetylcholine receptor.
abstract: In the absence of activating anionic lipids and cholesterol, the nicotinic acetylcholine receptor (nAChR) from Torpedo adopts an uncoupled conformation that does not usually gate open in response to agonist. The uncoupled conformation binds both agonists and non-competitive channel blockers with a lower affinity than the desensitized state, consistent with both the extracellular agonist-binding and transmembrane channel-gating domains individually adopting resting-state like conformations. To test this hypothesis, we characterized the binding of the agonist, acetylcholine, and two fluorescent channel blockers, ethidium and crystal violet, to resting, desensitized and uncoupled nAChRs in reconstituted membranes. The measured Kdfor acetylcholine binding to the uncoupled nAChR is similar to that for the resting state, confirming that the agonist binding site adopts a resting-state like conformation. Although both ethidium and crystal violet bind to the resting and desensitized channel pores with distinct affinities, no binding of either probe was detected to the uncoupled nAChR. Our data suggest that the transmembrane domain of the uncoupled nAChR adopts a conformation distinct from that of the resting and desensitized states. The lack of binding is consistent with a more constricted channel pore, possibly along the lines of what is observed in crystal structures of the prokaryotic homolog, ELIC.
authors.affiliation: Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Rd, K1H 8M5 Ottawa, ON, Canada.
authors.forename: Jiayin
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Rd, K1H 8M5 Ottawa, ON, Canada.
authors.forename: J Frederique
authors.initials: JF
authors.lastname: Comeau
authors.affiliation: Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Rd, K1H 8M5 Ottawa, ON, Canada. Electronic address: John.Baenziger@uottawa.ca.
authors.forename: John E
authors.initials: JE
authors.lastname: Baenziger
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D002784:Cholesterol / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014101:Torpedo / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D011978:Receptors, Nicotinic; D002784:Cholesterol; D000109:Acetylcholine
keywords: Channel gating; Channel pore; Lipid-protein interactions; Nicotinic acetylcholine receptor; Pentameric ligand-gated ion channels; Uncoupling