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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.


Related in: MedlinePlus

Effects of i.m. local injections of 20-meSPX-G in vivo on the mouse neuromuscular system. (A) Compound muscle action potentials (CMAPs) recorded from the tail muscle in response to caudal motor nerve stimulation (increasing intensities, scheme), before and at various times after injection of the cyclic imine toxin (35 pg/mouse); (B) time-course of the effects of 20-meSPX-G injections (1.75, 35 and 350.5 pg/mouse) on the CMAP maximal amplitude. Arrows indicate the time of injections; and (C) dose-response curve of the effects of 20-meSPX-G injections on the maximal CMAP amplitude. Each value represents the mean ± SEM of data obtained from 4–6 mice, and is expressed relatively to that obtained before injections. The sigmoid curve was obtained by nonlinear regression analysis through data points (R2 ≥ 0.99). The 20-meSPX-G dose required to block 50% of the maximal CMAP amplitude (dashed lines) was 47 pg/mouse.
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toxins-08-00249-f003: Effects of i.m. local injections of 20-meSPX-G in vivo on the mouse neuromuscular system. (A) Compound muscle action potentials (CMAPs) recorded from the tail muscle in response to caudal motor nerve stimulation (increasing intensities, scheme), before and at various times after injection of the cyclic imine toxin (35 pg/mouse); (B) time-course of the effects of 20-meSPX-G injections (1.75, 35 and 350.5 pg/mouse) on the CMAP maximal amplitude. Arrows indicate the time of injections; and (C) dose-response curve of the effects of 20-meSPX-G injections on the maximal CMAP amplitude. Each value represents the mean ± SEM of data obtained from 4–6 mice, and is expressed relatively to that obtained before injections. The sigmoid curve was obtained by nonlinear regression analysis through data points (R2 ≥ 0.99). The 20-meSPX-G dose required to block 50% of the maximal CMAP amplitude (dashed lines) was 47 pg/mouse.

Mentions: An in vivo study of the excitability properties of the mouse neuromuscular system after i.m. injection of 20-meSPX-G (from 1.75 to 350.5 pg/mouse) revealed that the major effect of the toxin was a noticeable time- and dose-dependent reduction of the compound muscle action potential (CMAP) amplitude in response to motor nerve stimulation, as shown in Figure 3A.


The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors
Effects of i.m. local injections of 20-meSPX-G in vivo on the mouse neuromuscular system. (A) Compound muscle action potentials (CMAPs) recorded from the tail muscle in response to caudal motor nerve stimulation (increasing intensities, scheme), before and at various times after injection of the cyclic imine toxin (35 pg/mouse); (B) time-course of the effects of 20-meSPX-G injections (1.75, 35 and 350.5 pg/mouse) on the CMAP maximal amplitude. Arrows indicate the time of injections; and (C) dose-response curve of the effects of 20-meSPX-G injections on the maximal CMAP amplitude. Each value represents the mean ± SEM of data obtained from 4–6 mice, and is expressed relatively to that obtained before injections. The sigmoid curve was obtained by nonlinear regression analysis through data points (R2 ≥ 0.99). The 20-meSPX-G dose required to block 50% of the maximal CMAP amplitude (dashed lines) was 47 pg/mouse.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-08-00249-f003: Effects of i.m. local injections of 20-meSPX-G in vivo on the mouse neuromuscular system. (A) Compound muscle action potentials (CMAPs) recorded from the tail muscle in response to caudal motor nerve stimulation (increasing intensities, scheme), before and at various times after injection of the cyclic imine toxin (35 pg/mouse); (B) time-course of the effects of 20-meSPX-G injections (1.75, 35 and 350.5 pg/mouse) on the CMAP maximal amplitude. Arrows indicate the time of injections; and (C) dose-response curve of the effects of 20-meSPX-G injections on the maximal CMAP amplitude. Each value represents the mean ± SEM of data obtained from 4–6 mice, and is expressed relatively to that obtained before injections. The sigmoid curve was obtained by nonlinear regression analysis through data points (R2 ≥ 0.99). The 20-meSPX-G dose required to block 50% of the maximal CMAP amplitude (dashed lines) was 47 pg/mouse.
Mentions: An in vivo study of the excitability properties of the mouse neuromuscular system after i.m. injection of 20-meSPX-G (from 1.75 to 350.5 pg/mouse) revealed that the major effect of the toxin was a noticeable time- and dose-dependent reduction of the compound muscle action potential (CMAP) amplitude in response to motor nerve stimulation, as shown in Figure 3A.

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.


Related in: MedlinePlus