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Toxins (Basel)
2016 Aug 24;89:. doi: 10.3390/toxins8090249.
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The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors.
Couesnon A
,
Aráoz R
,
Iorga BI
,
Benoit E
,
Reynaud M
,
Servent D
,
Molgó J
.
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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 [(125)I]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.
Figure 1. (A) Chemical structure of spirolide A toxin (the first member of the spirolide family described). The positions at which variations in the substitution pattern have been described are numbered, and the size of the macrocyclic is shown in magenta; (B) chemical structure of 20-methyl spirolide G.; (C) chemical structure of 13-desmethyl spirolide C; and (D) chemical structure of gymnodimine A.
Figure 2. (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.
Figure 3. 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.
Figure 4. Activation of Torpedo α12β1γδ nAChR incorporated into the oocyte membrane by an EC50 of ACh (25 µM), applied for 15 s duration (blue arrows), the inhibitory action of 3.12 nM 20-meSPX-G, and the washout of the spirolide from the medium. The two first inward nicotinic currents, recorded at −60 mV holding potential, correspond to the control ACh-evoked currents. The red tracing above the current trace denotes the perfusion of 20-meSPX-G before ACh perfusion (indicated by the blue arrow). No current was evoked by the perfusion of the spirolide alone, indicating that it has no direct agonist action on the receptors, while when ACh was applied in the presence of 20-meSPX-G (red arrow) a marked reduction in the ACh-evoked current occurred. The washout of 20-meSPX-G from the medium (indicated by a brown tracing below the current trace) only allowed partial recovery (≥50%) of control ACh-evoked currents.
Figure 5. 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).
Figure 6. 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).
Figure 7. 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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