title: Critical residues influence the affinity and selectivity of alpha-conotoxin MI for nicotinic acetylcholine receptors.
abstract: The mammalian skeletal muscle acetylcholine receptor contains two nonequivalent acetylcholine binding sites, one each at the alpha/delta and alpha/gamma subunit interfaces. Alpha-Conotoxin MI, a 14-amino acid competitive antagonist, binds at both interfaces but has approximately 10(4) higher affinity for the alpha/delta site. We performed an "alanine walk" to identify the residues in alpha-MI that contribute to this selective interaction with the alpha/delta site. Electrophysiological measurements with Xenopus oocytes expressing normal receptors or receptors lacking either the gamma or delta subunit were made to assay toxin-receptor interaction. Alanine substitutions in most amino acid positions had only modest effects on toxin potency at either binding site. However, substitutions in two positions, proline-6 and tyrosine-12, dramatically reduced toxin potency at the high-affinity alpha/delta site while having comparatively little effect on low-affinity alpha/gamma binding. When tyrosine-12 was replaced by alanine, the toxin's selectivity for the high-affinity site (relative to that for the low-affinity site) was reduced from 45,000- to 30-fold. A series of additional amino acid substitutions in this position showed that increasing side chain size/hydrophobicity increases toxin potency at the alpha/delta site without affecting alpha/gamma binding. In contrast, when tyrosine-12 is diiodinated, toxin binding is nearly irreversible at the alpha/delta site but also increases by approximately 500-fold at the alpha/gamma site. The effects of position 12 substitutions are accounted for almost entirely by changes in the rate of toxin dissociation from the high-affinity alpha/delta binding site.
authors.affiliation: Department of Biology, University of Utah, Salt Lake City 84112-0840, USA.
authors.forename: R B
authors.initials: RB
authors.lastname: Jacobsen
authors.forename: R G
authors.initials: RG
authors.lastname: DelaCruz
authors.forename: J H
authors.initials: JH
authors.lastname: Grose
authors.forename: J M
authors.initials: JM
authors.lastname: McIntosh
authors.forename: D
authors.initials: D
authors.lastname: Yoshikami
authors.forename: B M
authors.initials: BM
authors.lastname: Olivera
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020916:Conotoxins / Q000737:chemistry / Q000378:metabolism*; D004594:Electrophysiology; D051379:Mice; D008969:Molecular Sequence Data; D018733:Nicotinic Antagonists / Q000737:chemistry / Q000378:metabolism*; D009865:Oocytes / Q000737:chemistry / Q000502:physiology; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000378:metabolism; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014981:Xenopus
chemical_list: D020916:Conotoxins; D018733:Nicotinic Antagonists; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D011978:Receptors, Nicotinic; C036155:conotoxin MI; D000409:Alanine