title: Iodo-alpha-conotoxin MI selectively binds the alpha/delta subunit interface of muscle nicotinic acetylcholine receptors.
abstract: The embryonic mouse muscle nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel formed by alpha1, beta1, delta, and gamma subunits. The receptor contains two ligand binding sites at alpha/delta and alpha/gamma subunit interfaces. [(3)H]Curare preferentially binds the alpha/gamma interface. We describe the synthesis and properties of a high-affinity iodinated ligand that selectively binds the alpha/delta interface. An analogue of alpha-conotoxin MI was synthesized with an iodine attached to Tyr-12 (iodo-alpha-MI). The analogue potently blocks the fetal mouse muscle subtype of nAChR expressed in Xenopus oocytes. It failed, however, to block alpha3beta4, alpha4beta2, or alpha7 nAChRs. Iodo-alpha-MI potently blocks the alpha1beta1delta but not the alpha1beta1gamma subunit combination expressed in Xenopus oocytes indicating selectivity for the alpha/delta subunit interface. Alpha-conotoxin MI was subsequently radioiodinated, and its properties were further evaluated. Saturation experiments indicate that radioiodinated alpha-conotoxin MI binds to TE671 cell homogenates with a Hill slope of 0.95 +/- 0.0094. Kinetic studies indicate that the binding of [(125)I]alpha-conotoxin MI is reversible (k(off) = 0.084 +/- 0.0045 min(-1)); k(on) is 8.5 x 10(7) min(-1) M(-1). The calculated k(d) is 0.98 nM. This potency is approximately 20-fold higher than the unmodified alpha-MI peptide. Unlike [(125)I]alpha-bungarotoxin, [(125)I]alpha-conotoxin MI binding to TE671 cell homogenates is fully displaceable by the small molecule antagonist d-tubocurarine.
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.
authors.forename: Siqin
authors.initials: S
authors.lastname: Luo
authors.forename: J Michael
authors.initials: JM
authors.lastname: McIntosh
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D002478:Cells, Cultured; D020916:Conotoxins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D006801:Humans; D007455:Iodine / Q000737:chemistry; D007457:Iodine Radioisotopes; D007700:Kinetics; D051379:Mice; D018482:Muscle, Skeletal / Q000196:embryology / Q000378:metabolism; D018733:Nicotinic Antagonists / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014403:Tubocurarine / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry; D014981:Xenopus
chemical_list: D002038:Bungarotoxins; D020916:Conotoxins; D007457:Iodine Radioisotopes; D018733:Nicotinic Antagonists; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D014443:Tyrosine; C036155:conotoxin MI; D007455:Iodine; D014403:Tubocurarine