Conotoxin ad-GeXXA utilizes a novel strategy to antagonize nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) play essential roles in transmitting acetylcholine-mediated neural signals across synapses and neuromuscular junctions, and are also closely linked to various diseases and clinical conditions. Therefore, novel nAChR-specific compounds have great potential for both neuroscience research and clinical applications. Conotoxins, the peptide neurotoxins produced by cone snails, are a rich reservoir of novel ligands that target receptors, ion channels and transporters in the nervous system. From the venom of Conus generalis, we identified a novel dimeric nAChR-inhibiting alphaD-conotoxin GeXXA. By solving the crystal structure and performing structure-guided dissection of this toxin, we demonstrated that the monomeric C-terminal domain of alphaD-GeXXA, GeXXA-CTD, retains inhibitory activity against the alpha9alpha10 nAChR subtype. Furthermore, we identified that His7 of the rat alpha10 nAChR subunit determines the species preference of alphaD-GeXXA, and is probably part of the binding site of this toxin. These results together suggest that alphaD-GeXXA cooperatively binds to two inter-subunit interfaces on the top surface of nAChR, thus allosterically disturbing the opening of the receptor. The novel antagonistic mechanism of alphaD-GeXXA via a new binding site on nAChRs provides a valuable basis for the rational design of new nAChR-targeting compounds.