title: Acetylcholine promotes binding of α-conotoxin MII at α3 β2 nicotinic acetylcholine receptors.
abstract: α-Conotoxin MII (α-CTxMII) is a 16-residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2-Cys8 and Cys3-Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand-binding affinity, homology models of the heteropentameric α3β2-nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3- and β2-subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3β2-nAChR for α-CTxMII with ACh bound to the receptor, and this was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs.
authors.affiliation: Chemical and Biological Engineering Department, Colorado School of Mines, 1500 Illinois Street, Golden, CO 80401 (USA).
authors.forename: Somisetti V
authors.initials: SV
authors.lastname: Sambasivarao
authors.forename: Jessica
authors.initials: J
authors.lastname: Roberts
authors.forename: Vivek S
authors.initials: VS
authors.lastname: Bharadwaj
authors.forename: Jason G
authors.initials: JG
authors.lastname: Slingsby
authors.forename: Conrad
authors.initials: C
authors.lastname: Rohleder
authors.forename: Chris
authors.initials: C
authors.lastname: Mallory
authors.forename: James R
authors.initials: JR
authors.lastname: Groome
authors.forename: Owen M
authors.initials: OM
authors.lastname: McDougal
authors.forename: C Mark
authors.initials: CM
authors.lastname: Maupin
mesh_terms: D000109:Acetylcholine / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020916:Conotoxins / Q000737:chemistry / Q000378:metabolism*; D052078:Conus Snail / Q000378:metabolism; D030562:Databases, Protein; D007700:Kinetics; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D018733:Nicotinic Antagonists / Q000737:chemistry / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D055672:Static Electricity
chemical_list: D020916:Conotoxins; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; C098872:alpha-conotoxin MII; C419362:nicotinic receptor alpha3beta2; D000109:Acetylcholine
keywords: computer chemistry; conotoxins; homology modeling; receptors; two-electrode voltage clamp