Limits...
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

(A) Nerve-evoked isometric twitch responses on isolated mouse extensor digitorum longus (EDL) before and after the action of 10 nM 20-meSPX-G; and (B) concentration-response curve for the action of 20-meSPX-G on nerve-evoked isometric twitch response. Data points represent the normalized twitch response, relative to the respective controls. Each point is the mean ± SEM of 4 nerve muscle preparations at 60 min toxin exposure. The inset in B shows an example of superimposed twitch and tetanus response (40 Hz) triggered by direct electrical muscle stimulation when the nerve-evoked twitch was completely blocked by 10 nM 20-meSPX-G.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037475&req=5

toxins-08-00249-f002: (A) Nerve-evoked isometric twitch responses on isolated mouse extensor digitorum longus (EDL) before and after the action of 10 nM 20-meSPX-G; and (B) concentration-response curve for the action of 20-meSPX-G on nerve-evoked isometric twitch response. Data points represent the normalized twitch response, relative to the respective controls. Each point is the mean ± SEM of 4 nerve muscle preparations at 60 min toxin exposure. The inset in B shows an example of superimposed twitch and tetanus response (40 Hz) triggered by direct electrical muscle stimulation when the nerve-evoked twitch was completely blocked by 10 nM 20-meSPX-G.

Mentions: Exposure of isolated mouse extensor digitorum longus (EDL) neuromuscular preparations, endowed with the α12βδε nAChR, to the action of 20-meSPX-G revealed that the spirolide blocked in a time- and concentration-dependent manner the isometric twitch responses elicited by motor nerve stimulation (Figure 2). The onset of the contraction block was rapid at high 20-meSPX-G concentration, with a complete block of the twitch response within 60 min at concentrations above 10 nM.


The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors
(A) Nerve-evoked isometric twitch responses on isolated mouse extensor digitorum longus (EDL) before and after the action of 10 nM 20-meSPX-G; and (B) concentration-response curve for the action of 20-meSPX-G on nerve-evoked isometric twitch response. Data points represent the normalized twitch response, relative to the respective controls. Each point is the mean ± SEM of 4 nerve muscle preparations at 60 min toxin exposure. The inset in B shows an example of superimposed twitch and tetanus response (40 Hz) triggered by direct electrical muscle stimulation when the nerve-evoked twitch was completely blocked by 10 nM 20-meSPX-G.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-08-00249-f002: (A) Nerve-evoked isometric twitch responses on isolated mouse extensor digitorum longus (EDL) before and after the action of 10 nM 20-meSPX-G; and (B) concentration-response curve for the action of 20-meSPX-G on nerve-evoked isometric twitch response. Data points represent the normalized twitch response, relative to the respective controls. Each point is the mean ± SEM of 4 nerve muscle preparations at 60 min toxin exposure. The inset in B shows an example of superimposed twitch and tetanus response (40 Hz) triggered by direct electrical muscle stimulation when the nerve-evoked twitch was completely blocked by 10 nM 20-meSPX-G.
Mentions: Exposure of isolated mouse extensor digitorum longus (EDL) neuromuscular preparations, endowed with the α12βδε nAChR, to the action of 20-meSPX-G revealed that the spirolide blocked in a time- and concentration-dependent manner the isometric twitch responses elicited by motor nerve stimulation (Figure 2). The onset of the contraction block was rapid at high 20-meSPX-G concentration, with a complete block of the twitch response within 60 min at concentrations above 10 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.


Related in: MedlinePlus