title: Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor.
abstract: alpha-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. alpha-Conotoxins blocking muscle-type or alpha7 nAChRs compete with alpha-bungarotoxin. However, alpha-conotoxin ImII, a close homolog of the alpha7 nAChR-targeting alpha-conotoxin ImI, blocked alpha7 and muscle nAChRs without displacing alpha-bungarotoxin (Ellison et al. 2003, 2004), suggesting binding at a different site. We synthesized alpha-conotoxin ImII, its ribbon isomer (ImIIiso), 'mutant' ImII(W10Y) and found similar potencies in blocking human alpha7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [(125)I]-alpha-bungarotoxin from human alpha7 nAChRs in the cell line GH(4)C(1) (IC(50) 17 and 23 microM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC(50) 2.0-9.0 microM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized alpha-bungarotoxin (K(d) and IC(50) 2.5-8.2 microM). On Torpedo nAChR, alpha-conotoxin [(125)I]-ImII(W10Y) revealed specific binding (K(d) 1.5-6.1 microM) and could be displaced by alpha-conotoxin ImII, ImIIiso and ImII(W10Y) with IC(50) 2.7, 2.2 and 3.1 microM, respectively. As alpha-cobratoxin and alpha-conotoxin ImI displaced [(125)I]-ImII(W10Y) only at higher concentrations (IC(50)> or = 90 microM), our results indicate that alpha-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Igor E
authors.initials: IE
authors.lastname: Kasheverov
authors.forename: Maxim N
authors.initials: MN
authors.lastname: Zhmak
authors.forename: Alexander
authors.initials: A
authors.lastname: Fish
authors.forename: Prakash
authors.initials: P
authors.lastname: Rucktooa
authors.forename: Alexey Yu
authors.initials: AY
authors.lastname: Khruschov
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Osipov
authors.forename: Rustam H
authors.initials: RH
authors.lastname: Ziganshin
authors.forename: Dieter
authors.initials: D
authors.lastname: D'hoedt
authors.forename: Daniel
authors.initials: D
authors.lastname: Bertrand
authors.forename: Titia K
authors.initials: TK
authors.lastname: Sixma
authors.forename: August B
authors.initials: AB
authors.lastname: Smit
authors.forename: Victor I
authors.initials: VI
authors.lastname: Tsetlin
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects; D002038:Bungarotoxins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D020916:Conotoxins / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D007456:Iodine Isotopes / Q000378:metabolism; D008969:Molecular Sequence Data; D009865:Oocytes; D011869:Radioligand Assay / Q000379:methods; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D020349:Surface Plasmon Resonance / Q000379:methods; D014101:Torpedo / Q000378:metabolism*; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: D002038:Bungarotoxins; D002352:Carrier Proteins; C579167:Chrna7 protein, human; D020916:Conotoxins; D007456:Iodine Isotopes; D011978:Receptors, Nicotinic; C471178:alpha-conotoxin ImII, Conus imperialis; D064569:alpha7 Nicotinic Acetylcholine Receptor; C086863:Lymnaea furin2; D012697:Serine Endopeptidases; D000109:Acetylcholine