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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.


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.
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toxins-08-00249-f004: 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.

Mentions: Studies were carried out on Xenopus oocytes having incorporated to their membranes the Torpedo muscle-type α12β1γδ nAChR in order to determine whether 20-meSPX-G had an agonist and/or inhibitory action on this receptor subtype. For this, microtransplanted oocytes were voltage-clamped at −60 mV, and ACh was applied at the EC50 (25 µM) evoking typical inward nicotinic currents that exhibited similar amplitudes when delivered at 3 min interval and, upon washing, returned to the current basal line, as shown in a typical experiment (Figure 4). The fact that the amplitude of the ACh-evoked current was usually ≥1 µA (n = 6) was indicative of a good incorporation of the Torpedo muscle-type receptor into the oocyte membrane. However, when 3.1 nM 20-meSPX-G was applied to the same oocyte no change in the basal line current was observed, indicating that the toxin had no agonist action on the Torpedo muscle-type α12β1γδ nAChR (Figure 4).


The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors
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.
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Related In: Results  -  Collection

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toxins-08-00249-f004: 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.
Mentions: Studies were carried out on Xenopus oocytes having incorporated to their membranes the Torpedo muscle-type α12β1γδ nAChR in order to determine whether 20-meSPX-G had an agonist and/or inhibitory action on this receptor subtype. For this, microtransplanted oocytes were voltage-clamped at −60 mV, and ACh was applied at the EC50 (25 µM) evoking typical inward nicotinic currents that exhibited similar amplitudes when delivered at 3 min interval and, upon washing, returned to the current basal line, as shown in a typical experiment (Figure 4). The fact that the amplitude of the ACh-evoked current was usually ≥1 µA (n = 6) was indicative of a good incorporation of the Torpedo muscle-type receptor into the oocyte membrane. However, when 3.1 nM 20-meSPX-G was applied to the same oocyte no change in the basal line current was observed, indicating that the toxin had no agonist action on the Torpedo muscle-type α12β1γδ nAChR (Figure 4).

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.