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The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors

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ABSTRACT

The cyclic imine toxin 20-methyl spirolide G (20-meSPX-G), produced by the toxigenic dinoflagellate Alexandrium ostenfeldii/Alexandrium peruvianum, has been previously reported to contaminate shellfish in various European coastal locations, as revealed by mouse toxicity bioassay. The aim of the present study was to determine its toxicological profile and its molecular target selectivity. 20-meSPX-G blocked nerve-evoked isometric contractions in isolated mouse neuromuscular preparations, while it had no action on contractions elicited by direct electrical stimulation, and reduced reversibly nerve-evoked compound muscle action potential amplitudes in anesthetized mice. Voltage-clamp recordings in Xenopus oocytes revealed that 20-meSPX-G potently inhibited currents evoked by ACh on Torpedo muscle-type and human α7 nicotinic acetylcholine receptors (nAChR), whereas lower potency was observed in human α4β2 nAChR. Competition-binding assays showed that 20-meSPX-G fully displaced [3H]epibatidine binding to HEK-293 cells expressing the human α3β2 (Ki = 0.040 nM), whereas a 90-fold lower affinity was detected in human α4β2 nAChR. The spirolide displaced [125I]α-bungarotoxin binding to Torpedo membranes (Ki = 0.028 nM) and in HEK-293 cells expressing chick chimeric α7-5HT3 nAChR (Ki = 0.11 nM). In conclusion, this is the first study to demonstrate that 20-meSPX-G is a potent antagonist of nAChRs, and its subtype selectivity is discussed on the basis of molecular docking models.

No MeSH data available.


Protein-ligand interactions in the docking complexes of 20-meSPX-G (green) with four nAChR subtypes. (A) human α7 (orange, α7-α7 interface); (B) human α4β2 (yellow, α4-β2 interface); (C) human α3β2 (cyan, α3-β2 interface); and (D) Torpedo α12β1γδ (light blue, α1-δ interface). Only amino acids interacting through hydrogen bonds with the ligand or involved in toxin’s subtype selectivity, and in the sequence alignment, are shown. The numbering of amino acid residues is the same as in [21].
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toxins-08-00249-f007: Protein-ligand interactions in the docking complexes of 20-meSPX-G (green) with four nAChR subtypes. (A) human α7 (orange, α7-α7 interface); (B) human α4β2 (yellow, α4-β2 interface); (C) human α3β2 (cyan, α3-β2 interface); and (D) Torpedo α12β1γδ (light blue, α1-δ interface). Only amino acids interacting through hydrogen bonds with the ligand or involved in toxin’s subtype selectivity, and in the sequence alignment, are shown. The numbering of amino acid residues is the same as in [21].

Mentions: Molecular docking calculations were performed to evaluate the interactions of 20-meSPX-G with the extracellular domain of the four nAChR subtypes studied (human α7, α4β2, α3β2, and α12β1γδ). The receptor-ligand complexes (Figure 7) show binding modes similar to those observed in our previous studies, which were focused on the interaction between pinnatoxin A [24], 13-desmethyl spirolide C, and 13,19-didesmethyl spirolide C [23], and different nAChR subtypes.


The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors
Protein-ligand interactions in the docking complexes of 20-meSPX-G (green) with four nAChR subtypes. (A) human α7 (orange, α7-α7 interface); (B) human α4β2 (yellow, α4-β2 interface); (C) human α3β2 (cyan, α3-β2 interface); and (D) Torpedo α12β1γδ (light blue, α1-δ interface). Only amino acids interacting through hydrogen bonds with the ligand or involved in toxin’s subtype selectivity, and in the sequence alignment, are shown. The numbering of amino acid residues is the same as in [21].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037475&req=5

toxins-08-00249-f007: Protein-ligand interactions in the docking complexes of 20-meSPX-G (green) with four nAChR subtypes. (A) human α7 (orange, α7-α7 interface); (B) human α4β2 (yellow, α4-β2 interface); (C) human α3β2 (cyan, α3-β2 interface); and (D) Torpedo α12β1γδ (light blue, α1-δ interface). Only amino acids interacting through hydrogen bonds with the ligand or involved in toxin’s subtype selectivity, and in the sequence alignment, are shown. The numbering of amino acid residues is the same as in [21].
Mentions: Molecular docking calculations were performed to evaluate the interactions of 20-meSPX-G with the extracellular domain of the four nAChR subtypes studied (human α7, α4β2, α3β2, and α12β1γδ). The receptor-ligand complexes (Figure 7) show binding modes similar to those observed in our previous studies, which were focused on the interaction between pinnatoxin A [24], 13-desmethyl spirolide C, and 13,19-didesmethyl spirolide C [23], and different nAChR subtypes.

View Article: PubMed Central - PubMed

ABSTRACT

The cyclic imine toxin 20-methyl spirolide G (20-meSPX-G), produced by the toxigenic dinoflagellate Alexandrium ostenfeldii/Alexandrium peruvianum, has been previously reported to contaminate shellfish in various European coastal locations, as revealed by mouse toxicity bioassay. The aim of the present study was to determine its toxicological profile and its molecular target selectivity. 20-meSPX-G blocked nerve-evoked isometric contractions in isolated mouse neuromuscular preparations, while it had no action on contractions elicited by direct electrical stimulation, and reduced reversibly nerve-evoked compound muscle action potential amplitudes in anesthetized mice. Voltage-clamp recordings in Xenopus oocytes revealed that 20-meSPX-G potently inhibited currents evoked by ACh on Torpedo muscle-type and human α7 nicotinic acetylcholine receptors (nAChR), whereas lower potency was observed in human α4β2 nAChR. Competition-binding assays showed that 20-meSPX-G fully displaced [3H]epibatidine binding to HEK-293 cells expressing the human α3β2 (Ki = 0.040 nM), whereas a 90-fold lower affinity was detected in human α4β2 nAChR. The spirolide displaced [125I]α-bungarotoxin binding to Torpedo membranes (Ki = 0.028 nM) and in HEK-293 cells expressing chick chimeric α7-5HT3 nAChR (Ki = 0.11 nM). In conclusion, this is the first study to demonstrate that 20-meSPX-G is a potent antagonist of nAChRs, and its subtype selectivity is discussed on the basis of molecular docking models.

No MeSH data available.