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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 (I_ACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. αM-MIIIJ also protected against slowly-reversible block of I_ACh 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.

SUBMITTER: Rybin MJ

PROVIDER: S-EPMC7150935 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Ribbon ?-Conotoxin KTM Exhibits Potent Inhibition of Nicotinic Acetylcholine Receptors.

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Fluorescently Labeled α-Conotoxin TxID, a New Probe for α3β4 Neuronal Nicotinic Acetylcholine Receptors.

Project description:Neuronal nicotinic acetylcholine receptors (nAChRs) are important ion channel membrane proteins that are widely distributed in the central nervous system (CNS) and peripheral nervous system (PNS). As an important member, α3β4 nAChRs are related to pain sensation in PNS and nicotine addiction in CNS. However, research related to the α3β4 nAChRs is greatly limited by the lack of subtype-selective pharmacological tools. The α-conotoxin (α-CTx) TxID from the marine cone snail, Conus textile, is a selective α3β4 nAChR antagonist with relatively high potency. In this study, a fluorescent dye (5-TAMRA SE) was used to label TxID on the N-terminus of α-CTx TxID, and pure TxID-F (fluorescent analogue of TxID) was obtained by HPLC. At the same time, the potency and selectivity of TxID-F were detected by high-performance liquid chromatography (HPLC). Additionally, the potency and selectivity of TxID-F were determined by using a two-electrode voltage-clamp technique on various nAChRs expressed in the Xenopus oocyte expression system. The results obtained by electrophysiology showed that TxID-F maintained the same order of potency (IC50 73 nM) as the native toxin (IC50 25 nM) for the α3β4 nAChR subtype. In addition, the results of fluorescent spectroscopy and circular dichroism showed TxID-F has the same fluorescence as 5-TAMRA SE, as well as similar profiles as TxID. The results of flow cytometry showed that the histogram shifted significantly to the right for the RAW264.7 cells expressing α3β4-containing nAChRs stained with TxID-F and confirmed by live cell imaging. The study of fluorescent-labeled α-CTx TxID provides a rich pharmacological tool to explore the structure-function relationship, distribution, and ligand-binding domain of α3β4 nAChR subtype in the future.

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