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

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.


Dose-dependent inhibition of [125I]α-BTX binding on Torpedo or α7-5HT3 receptors by 20-meSPX-G, α-toxin, and methyllycaconitine (MLA) (A) and dose-dependent inhibition of [3H]epibatidine binding on α3β2 and α4β2 receptors with 20-meSPX-G and epibatidine (B).
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toxins-08-00249-f006: Dose-dependent inhibition of [125I]α-BTX binding on Torpedo or α7-5HT3 receptors by 20-meSPX-G, α-toxin, and methyllycaconitine (MLA) (A) and dose-dependent inhibition of [3H]epibatidine binding on α3β2 and α4β2 receptors with 20-meSPX-G and epibatidine (B).

Mentions: To obtain further information on the interaction between 20-meSPX-G and nAChR subtypes concerning their nAChR selectivity profile, binding affinities, and antagonist potencies, competition-binding assays were carried out with purified Torpedo membranes expressing muscle-type (α12βγδ) nAChR, and in HEK-293 cells expressing the chimeric chick neuronal α7-5HT3, and the human α4β2, and α3β2 nAChR subtypes using both [125I]α-bungarotoxin ([125I]α-BTX) and [3H]epibatidine as radiotracers. As shown in Figure 6A, 20-meSPX-G concentration-dependently displaced [125I]α-BTX from Torpedo membranes expressing the muscle-type nAChR, and from HEK-293 cells expressing the chimeric α7-5HT3 neuronal nAChR. Furthermore, 20-meSPX-G, in the range of concentrations studied, fully displaced [3H]epibatidine binding to HEK-293 cells expressing the human α3β2 and α4β2 subtypes (Figure 6B). The affinity constants computed from the binding-competition curves clearly show that 20-meSPX-G interacts with sub-nanomolar affinity with the muscle-type, α3β2, and α7-5HT3 nAChRs. On these receptors, 20-meSPX-G was as effective as high-affinity reference-standards, such as the snake α-toxin for skeletal muscle nAChR, methyllycaconitine (MLA) for α7 or chimeric α7-5HT3, and epibatidine for α3β2 nAChR subtypes. As reported on Table 1, the 20-meSPX-G interacted less efficiently with the α4β2 nAChR subtype with a Ki = 3.6 nM.


The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors
Dose-dependent inhibition of [125I]α-BTX binding on Torpedo or α7-5HT3 receptors by 20-meSPX-G, α-toxin, and methyllycaconitine (MLA) (A) and dose-dependent inhibition of [3H]epibatidine binding on α3β2 and α4β2 receptors with 20-meSPX-G and epibatidine (B).
© Copyright Policy
Related In: Results  -  Collection

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

toxins-08-00249-f006: Dose-dependent inhibition of [125I]α-BTX binding on Torpedo or α7-5HT3 receptors by 20-meSPX-G, α-toxin, and methyllycaconitine (MLA) (A) and dose-dependent inhibition of [3H]epibatidine binding on α3β2 and α4β2 receptors with 20-meSPX-G and epibatidine (B).
Mentions: To obtain further information on the interaction between 20-meSPX-G and nAChR subtypes concerning their nAChR selectivity profile, binding affinities, and antagonist potencies, competition-binding assays were carried out with purified Torpedo membranes expressing muscle-type (α12βγδ) nAChR, and in HEK-293 cells expressing the chimeric chick neuronal α7-5HT3, and the human α4β2, and α3β2 nAChR subtypes using both [125I]α-bungarotoxin ([125I]α-BTX) and [3H]epibatidine as radiotracers. As shown in Figure 6A, 20-meSPX-G concentration-dependently displaced [125I]α-BTX from Torpedo membranes expressing the muscle-type nAChR, and from HEK-293 cells expressing the chimeric α7-5HT3 neuronal nAChR. Furthermore, 20-meSPX-G, in the range of concentrations studied, fully displaced [3H]epibatidine binding to HEK-293 cells expressing the human α3β2 and α4β2 subtypes (Figure 6B). The affinity constants computed from the binding-competition curves clearly show that 20-meSPX-G interacts with sub-nanomolar affinity with the muscle-type, α3β2, and α7-5HT3 nAChRs. On these receptors, 20-meSPX-G was as effective as high-affinity reference-standards, such as the snake α-toxin for skeletal muscle nAChR, methyllycaconitine (MLA) for α7 or chimeric α7-5HT3, and epibatidine for α3β2 nAChR subtypes. As reported on Table 1, the 20-meSPX-G interacted less efficiently with the α4β2 nAChR subtype with a Ki = 3.6 nM.

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.