Ω-NEUROTOXINS: NOVEL ANTAGONISTS OF NICOTINIC ACETYLCHOLINE RECEPTORS FROM SNAKE VENOM

Abstract

Snake venom a-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: O-neurotoxins. Although they belong to the 3FTx family, the characteristic functional residues of a-neurotoxins are not conserved. We evaluated the subtype specificity and structure?function relationships of Oh9-1, an O-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 a1?1ed (adult), and a1?1?d (fetal) and rat neuronal a3?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 a1?1ed and a3?2 subtypes. Oh9-1?s loop-II residues (M25, F27) were the most critical for interactions and formed the common binding core. Mutations at T23 and F26 caused significant loss in activity at a1?1ed receptors, but had no effect on the interaction with the a3?2 subtype. Similarly, mutations at loop-II (H7, K22, H30) and -III (K45) of Oh9-1 had distinctly different impact on its activity with these subtypes. Thus, Oh9-1 interacts with these nAChRs via distinct residues. Unlike a-neurotoxins, the tip of loop-II is not involved. We reveal a novel mode of interaction, where both sides of the ?-strand of Oh9-1?s loop-II interact with a1?1ed, but only one side interacts with a3?2. Phylogenetic analysis revealed functional organisation of the O-neurotoxins independent of a-neurotoxins. Thus, O-neurotoxin Oh9-1 may be a new, structurally distinct class of 3FTxs that, like a-neurotoxins, antagonize nAChRs. However, Oh9-1 binds to the ACh binding pocket via a different set of functional residues.