Acetylcholine promotes binding of α-conotoxin MII at α3 β2 nicotinic acetylcholine receptors.

alpha-Conotoxin MII (alpha-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 alpha(3)beta(2)-nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorat alpha-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californic alpha-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the alpha(3)- and beta(2)-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 alpha-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 alpha-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the alpha(3)beta(2)-nAChR for alpha-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 alpha-CTxMIIs on nAChRs.