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

Abstract

We report the discovery and functional characterization of αM-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail Conus magus. Injections of αM-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 αM-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC50 of ~0.1 μM. With comparable potency, αM-MIIIJ reversibly blocked ACh-gated currents (IACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. αM-MIIIJ also protected against slowly-reversible block of IACh by α-bungarotoxin (α-BgTX, a snake neurotoxin) and α-conotoxin EI (α-EI, from Conus ermineus another fish hunter) that competitively block nAChRs at the ACh binding site. Furthermore, assessment by fluorescence microscopy showed that αM-MIIIJ inhibited the binding of fluorescently-tagged α-BgTX at neuromuscular junctions of X. laevis, C. auratus, and D. rerio. (Note, we observed that αM-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 αM-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 αM-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.

Marine natural products

“…668 Functional investigation of aM-conotoxin MIIIJ (C. magus) has revealed it to be an inhibitor of muscular nAChRs by interfering with acetylcholine binding. 669 The a9a10 nAChR antagonist aO-conotoxin GeXIVA induces cell cycle arrest and apoptosis in the MDA-MB-157 breast cancer cell line, 670,671 while cytotoxic synergism was observed for combinations of a-conotoxins, including PnIA, RgIA, MII, with lipoxygenase and cyclooxygenase inhibitors towards Ehrlich carcinoma cells in vitro. 672 Disulde-decient analogues of a-conotoxin GeXIVA retain some measure of a9a10 nAChR antagonistic activities, with NMR and molecular dynamics simulations suggesting the disulde bonds and peptide termini do interact with the receptor and that the central segment of residues 12 to 18 can form a stable a-helical conformation that binds to the receptor.…”

Discovery of a Potent Conorfamide from Conus episcopatus Using a Novel Zebrafish Larvae Assay

“…Zebrafish are also emerging as a useful model system to study the biological functions of conotoxins. Previous studies established that treatment with different conotoxins could elicit behavioral changes in fish. , Many in vivo models used to study these conotoxins require the use of injections which can greatly reduce the potential throughput of these methods, but as demonstrated recently, the addition of venom insulin Con-Insulin G1 and conotoxin ρ-TIa directly into the water is sufficient to alter zebrafish behavior, thus removing the need for injection.…”

Marine Origin Ligands of Nicotinic Receptors: Low Molecular Compounds, Peptides and Proteins for Fundamental Research and Practical Applications

“…The last decade has supplemented this list with two new families: αB [ 99 ] and αO-conotoxins [ 100 ] (see Figure 2 ); however, these families are represented by one or only a few peptides, which, as a rule, have not received further scientific development. As an exception, we can mention the discovery of a new αS-conotoxin, GVIIIB, which was a highly selective inhibitor of α9α10 nAChR [ 101 ], αM-conotoxin MIIIJ, which, like ψ-conotoxins, belongs to the M-superfamily and also blocks muscle nAChR [ 102 ], αO-conotoxin GeXIVA [ 100 ], with good prospects for medical use (see below) and several αD-conotoxins. Of the two isolated dimeric αD-conotoxins, GeXXA [ 103 ] ( Figure 3 ) and PiXXA [ 104 ], the first was shown to be a non-selective blocker of both neuronal and muscle receptor subtypes, while for the second, inhibitory activity was revealed only towards human α7 nAChR (IC 50 6.2 µM).…”