Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

alpha-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. alpha-Conotoxins blocking muscle-type or alpha 7 nAChRs compete with alpha-bungarotoxin. However, alpha-conotoxin ImII, a close homolog of the alpha 7 nAChR-targeting alpha-conotoxin ImI, blocked alpha 7 and muscle nAChRs without displacing alpha-bungarotoxin (Ellison et al. 2003, 2004), suggesting binding at a different site. We synthesized alpha-conotoxin ImII, its ribbon isomer (ImIIiso), 'mutant' ImII(W10Y) and found similar potencies in blocking human alpha 7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [125I]-alpha-bungarotoxin from human alpha 7 nAChRs in the cell line GH(4)C(1) (IC50 17 and 23 mu M, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC50 2.0-9.0 mu M). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized alpha-bungarotoxin (K-d and IC50 2.5-8.2 mu M). On Torpedo nAChR, alpha-conotoxin [125I]-ImII(W10Y) revealed specific binding (K-d 1.5-6.1 mu M) and could be displaced by alpha-conotoxin ImII, ImIIiso and ImII(W10Y) with IC50 2.7, 2.2 and 3.1 mu M, respectively. As alpha-cobratoxin and alpha-conotoxin ImI displaced [125I]-ImII(W10Y) only at higher concentrations (IC50 >= 90 mu M), our results indicate that alpha-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.