alphaM-Conotoxin MIIIJ Blocks Nicotinic Acetylcholine Receptors at Neuromuscular Junctions of Frog and Fish.

We report the discovery and functional characterization of alphaM-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail Conus magus. Injections of alphaM-MIIIJ induced paralysis in goldfish (Carassius auratus) but not mice. Intracellular recording from skeletal muscles of fish (C. auratus) and frog (Xenopus laevis) revealed that alphaM-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC50 of ~0.1 muM. With comparable potency, alphaM-MIIIJ reversibly blocked ACh-gated currents (IACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. alphaM-MIIIJ also protected against slowly-reversible block of IACh by alpha-bungarotoxin (alpha-BgTX, a snake neurotoxin) and alpha-conotoxin EI (alpha-EI, from Conus ermineus another fish hunter) that competitively block nAChRs at the ACh binding site. Furthermore, assessment by fluorescence microscopy showed that alphaM-MIIIJ inhibited the binding of fluorescently-tagged alpha-BgTX at neuromuscular junctions of X. laevis, C. auratus, and D. rerio. (Note, we observed that alphaM-MIIIJ can block adult mouse and human muscle nAChRs exogenously expressed in X. laevis oocytes, but with IC50s ~100-times higher than those of zebrafish nAChRs.) Taken together, these results indicate that alphaM-MIIIJ inhibits muscle nAChRs and furthermore apparently does so by interfering with the binding of ACh to its receptor. Comparative alignments with homologous sequences identified in other fish hunters revealed that alphaM-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.