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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.

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Concentration-dependent inhibition of ACh-elicited nicotinic currents by 20-meSPX-G in Torpedo α12β1γδ (blue curve); human α7 (red curve); and human α4β2 (orange curve) nAChRs incorporated or expressed in Xenopus oocytes. Peak amplitudes of ACh-evoked currents (mean ± SEM), were recorded under voltage-clamp conditions at −60 mV holding membrane potential, in the presence of 20-meSPX-G were normalized to control currents and fitted to the Hill equation. The concentration of ACh used for each nAChR subtype was the EC50 experimentally determined (see text for details).
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toxins-08-00249-f005: Concentration-dependent inhibition of ACh-elicited nicotinic currents by 20-meSPX-G in Torpedo α12β1γδ (blue curve); human α7 (red curve); and human α4β2 (orange curve) nAChRs incorporated or expressed in Xenopus oocytes. Peak amplitudes of ACh-evoked currents (mean ± SEM), were recorded under voltage-clamp conditions at −60 mV holding membrane potential, in the presence of 20-meSPX-G were normalized to control currents and fitted to the Hill equation. The concentration of ACh used for each nAChR subtype was the EC50 experimentally determined (see text for details).

Mentions: In contrast, when ACh was applied together with 3.1 nM 20-meSPX-G a marked block of the inward nicotinic current was observed, as shown in Figure 4. This blockade of nAChR was persistent, as determined by washing out 20-meSPX-G from the extracellular medium and delivering a constant ACh concentration spaced by a 3 min interval, since the recovery of the ACh-evoked current did not attain 50% within a 60 min period (Figure 4). These results suggest that 20-meSPX-G has a slow Koff from the Torpedo α12β1γδ nAChR subtype. As shown in Figure 5, the inhibitory action of 20-meSPX-G on the muscle-type receptor was concentration-dependent, with an IC50 = 0.36 nM (0.29−0.45 nM, 95% confidence intervals, 50 oocytes from 8 Xenopus donors).


The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors
Concentration-dependent inhibition of ACh-elicited nicotinic currents by 20-meSPX-G in Torpedo α12β1γδ (blue curve); human α7 (red curve); and human α4β2 (orange curve) nAChRs incorporated or expressed in Xenopus oocytes. Peak amplitudes of ACh-evoked currents (mean ± SEM), were recorded under voltage-clamp conditions at −60 mV holding membrane potential, in the presence of 20-meSPX-G were normalized to control currents and fitted to the Hill equation. The concentration of ACh used for each nAChR subtype was the EC50 experimentally determined (see text for details).
© Copyright Policy
Related In: Results  -  Collection

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

toxins-08-00249-f005: Concentration-dependent inhibition of ACh-elicited nicotinic currents by 20-meSPX-G in Torpedo α12β1γδ (blue curve); human α7 (red curve); and human α4β2 (orange curve) nAChRs incorporated or expressed in Xenopus oocytes. Peak amplitudes of ACh-evoked currents (mean ± SEM), were recorded under voltage-clamp conditions at −60 mV holding membrane potential, in the presence of 20-meSPX-G were normalized to control currents and fitted to the Hill equation. The concentration of ACh used for each nAChR subtype was the EC50 experimentally determined (see text for details).
Mentions: In contrast, when ACh was applied together with 3.1 nM 20-meSPX-G a marked block of the inward nicotinic current was observed, as shown in Figure 4. This blockade of nAChR was persistent, as determined by washing out 20-meSPX-G from the extracellular medium and delivering a constant ACh concentration spaced by a 3 min interval, since the recovery of the ACh-evoked current did not attain 50% within a 60 min period (Figure 4). These results suggest that 20-meSPX-G has a slow Koff from the Torpedo α12β1γδ nAChR subtype. As shown in Figure 5, the inhibitory action of 20-meSPX-G on the muscle-type receptor was concentration-dependent, with an IC50 = 0.36 nM (0.29−0.45 nM, 95% confidence intervals, 50 oocytes from 8 Xenopus donors).

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