文摘
Snake venom 伪-neurotoxins from the three-finger toxin (3FTx) family are competitive antagonists with nanomolar affinity and high selectivity for nicotinic acetylcholine receptors (nAChR). Here, we report the characterization of a new group of competitive nAChR antagonists: 惟-neurotoxins. Although they belong to the 3FTx family, the characteristic functional residues of 伪-neurotoxins are not conserved. We evaluated the subtype specificity and structure鈥揻unction relationships of Oh9-1, an 惟-neurotoxin from Ophiophagus hannah venom. Recombinant Oh9-1 showed reversible postsynaptic neurotoxicity in the micromolar range. Experiments with different nAChR subtypes expressed in Xenopus oocytes indicated Oh9-1 is selective for rat muscle type 伪1尾1蔚未 (adult) and 伪1尾1纬未 (fetal) and rat neuronal 伪3尾2 subtypes. However, Oh9-1 showed low or no affinity for other human and rat neuronal subtypes. Twelve individual alanine-scan mutants encompassing all three loops of Oh9-1 were evaluated for binding to 伪1尾1蔚未 and 伪3尾2 subtypes. Oh9-1鈥檚 loop-II residues (M25, F27) were the most critical for interactions and formed the common binding core. Mutations at T23 and F26 caused a significant loss in activity at 伪1尾1蔚未 receptors but had no effect on the interaction with the 伪3尾2 subtype. Similarly, mutations at loop-II (H7, K22, H30) and -III (K45) of Oh9-1 had a distinctly different impact on its activity with these subtypes. Thus, Oh9-1 interacts with these nAChRs via distinct residues. Unlike 伪-neurotoxins, the tip of loop-II is not involved. We reveal a novel mode of interaction, where both sides of the 尾-strand of Oh9-1鈥檚 loop-II interact with 伪1尾1蔚未, but only one side interacts with 伪3尾2. Phylogenetic analysis revealed functional organization of the 惟-neurotoxins independent of 伪-neurotoxins. Thus, 惟-neurotoxin: Oh9-1 may be a new, structurally distinct class of 3FTxs that, like 伪-neurotoxins, antagonize nAChRs. However, Oh9-1 binds to the ACh binding pocket via a different set of functional residues.