TY - JOUR

T1 - alpha M-Conotoxin MIIIJ Blocks Nicotinic Acetylcholine Receptors at Neuromuscular Junctions of Frog and Fish

AU - Rybin, Matthew J.

AU - O'Brien, Henrik

AU - Ramiro, Iris Bea L.

AU - Azam, Layla

AU - McIntosh, J. Michael

AU - Olivera, Baldomero M.

AU - Safavi-Hemami, Helena

AU - Yoshikami, Doju

PY - 2020

Y1 - 2020

N2 - We report the discovery and functional characterization of alpha M-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail Conus magus. Injections of alpha M-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 alpha M-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC50 of similar to 0.1 mu M. With comparable potency, alpha M-MIIIJ reversibly blocked ACh-gated currents (I-ACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. alpha M-MIIIJ also protected against slowly-reversible block of I-ACh 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 alpha M-MIIIJ inhibited the binding of fluorescently-tagged alpha-BgTX at neuromuscular junctions of X. laevis, C. auratus, and D. rerio. (Note, we observed that alpha M-MIIIJ can block adult mouse and human muscle nAChRs exogenously expressed in X. laevis oocytes, but with IC(50)s similar to 100-times higher than those of zebrafish nAChRs.) Taken together, these results indicate that alpha M-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 alpha M-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.

AB - We report the discovery and functional characterization of alpha M-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail Conus magus. Injections of alpha M-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 alpha M-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC50 of similar to 0.1 mu M. With comparable potency, alpha M-MIIIJ reversibly blocked ACh-gated currents (I-ACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. alpha M-MIIIJ also protected against slowly-reversible block of I-ACh 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 alpha M-MIIIJ inhibited the binding of fluorescently-tagged alpha-BgTX at neuromuscular junctions of X. laevis, C. auratus, and D. rerio. (Note, we observed that alpha M-MIIIJ can block adult mouse and human muscle nAChRs exogenously expressed in X. laevis oocytes, but with IC(50)s similar to 100-times higher than those of zebrafish nAChRs.) Taken together, these results indicate that alpha M-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 alpha M-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.

KW - venom

KW - conotoxin

KW - nicotinic acetylcholine receptor

KW - neuromuscular junction

KW - SODIUM-CHANNELS

KW - CONUS-GEOGRAPHUS

KW - PREFERENTIAL INHIBITION

KW - OMEGA-CONOTOXIN

KW - SKELETAL-MUSCLE

KW - VENOM PEPTIDES

KW - PSI-CONOTOXIN

KW - TRANSMISSION

KW - ANTAGONIST

KW - TARGETS

U2 - 10.3390/toxins12030197

DO - 10.3390/toxins12030197

M3 - Journal article

C2 - 32245200

VL - 12

JO - Toxins

JF - Toxins

SN - 2072-6651

IS - 3

M1 - 197

ER -