title: Water-soluble LYNX1 residues important for interaction with muscle-type and/or neuronal nicotinic receptors.
abstract: Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the α7 nAChR extracellular ligand-binding domain and the transmembrane domain of α1 glycine receptor (α7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at α7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on α7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for α-bungarotoxin and similar snake α-neurotoxins also targeting α7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997 Moscow, Russia.
authors.forename: Ekaterina N
authors.initials: EN
authors.lastname: Lyukmanova
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Shulepko
authors.forename: Svetlana L
authors.initials: SL
authors.lastname: Buldakova
authors.forename: Igor E
authors.initials: IE
authors.lastname: Kasheverov
authors.forename: Zakhar O
authors.initials: ZO
authors.lastname: Shenkarev
authors.forename: Roman V
authors.initials: RV
authors.lastname: Reshetnikov
authors.forename: Sergey Y
authors.initials: SY
authors.lastname: Filkin
authors.forename: Denis S
authors.initials: DS
authors.lastname: Kudryavtsev
authors.forename: Lucy O
authors.initials: LO
authors.lastname: Ojomoko
authors.forename: Elena V
authors.initials: EV
authors.lastname: Kryukova
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: Mikhail P
authors.initials: MP
authors.lastname: Kirpichnikov
authors.forename: Piotr D
authors.initials: PD
authors.lastname: Bregestovski
authors.forename: Victor I
authors.initials: VI
authors.lastname: Tsetlin
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D000818:Animals; D002038:Bungarotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058851:GPI-Linked Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014101:Torpedo; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002038:Bungarotoxins; C579167:Chrna7 protein, human; D029941:Fish Proteins; D058851:GPI-Linked Proteins; C485626:LYNX1 protein, human; D009498:Neurotoxins; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor
keywords: Acetylcholine-binding Proteins; Computer Modeling; Electrophysiology; Ionic Currents; LYNX1; Mutagenesis; Neurotoxin; Nicotinic Acetylcholine Receptors; Radioligand Assay