title: The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors.
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 [(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.
authors: Institut des Neurosciences Paris-Saclay, UMR 9197 CNRS/Université Paris-Sud, F-91190 Gif-sur-Yvette, France. aurelie.couesnon@inaf.cnrs-gif.fr.
authors: Aurélie
authors: A
authors: Couesnon
authors: Institut des Neurosciences Paris-Saclay, UMR 9197 CNRS/Université Paris-Sud, F-91190 Gif-sur-Yvette, France. romulo.araoz@cea.fr.|Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie et Technologies de Saclay (IBITECS), Université Paris-Saclay, Service d'Ingénierie Moléculaire des Protéines, bâtiment 152, F-91191 Gif-sur-Yvette, France. romulo.araoz@cea.fr.
authors: Rómulo
authors: R
authors: Aráoz
authors: Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles, UPR 2301, Labex LERMIT, F-91198 Gif-sur-Yvette, France. bogdan.iorga@cnrs.fr.
authors: Bogdan I
authors: BI
authors: Iorga
authors: Institut des Neurosciences Paris-Saclay, UMR 9197 CNRS/Université Paris-Sud, F-91190 Gif-sur-Yvette, France. evelyne.benoit@cea.fr.|Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie et Technologies de Saclay (IBITECS), Université Paris-Saclay, Service d'Ingénierie Moléculaire des Protéines, bâtiment 152, F-91191 Gif-sur-Yvette, France. evelyne.benoit@cea.fr.
authors: Evelyne
authors: E
authors: Benoit
authors: Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie et Technologies de Saclay (IBITECS), Université Paris-Saclay, Service d'Ingénierie Moléculaire des Protéines, bâtiment 152, F-91191 Gif-sur-Yvette, France. morgane.reynaud@hotmail.fr.
authors: Morgane
authors: M
authors: Reynaud
authors: Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie et Technologies de Saclay (IBITECS), Université Paris-Saclay, Service d'Ingénierie Moléculaire des Protéines, bâtiment 152, F-91191 Gif-sur-Yvette, France. denis.servent@cea.fr.
authors: Denis
authors: D
authors: Servent
authors: Institut des Neurosciences Paris-Saclay, UMR 9197 CNRS/Université Paris-Sud, F-91190 Gif-sur-Yvette, France. jordi.molgo@cea.fr.|Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie et Technologies de Saclay (IBITECS), Université Paris-Saclay, Service d'Ingénierie Moléculaire des Protéines, bâtiment 152, F-91191 Gif-sur-Yvette, France. jordi.molgo@cea.fr.
authors: Jordi
authors: J
authors: Molgó
mesh_terms: D000200:Action Potentials; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002645:Chickens; D002799:Cholinergic Fibers / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects*; D051379:Mice; D062105:Molecular Docking Simulation; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000294:innervation / Q000378:metabolism; D009469:Neuromuscular Junction / Q000187:drug effects* / Q000378:metabolism; D018733:Nicotinic Antagonists / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D011485:Protein Binding; D011487:Protein Conformation; D011725:Pyridines / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013141:Spiro Compounds / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D013329:Structure-Activity Relationship; D014101:Torpedo; D014162:Transfection; D014982:Xenopus laevis
chemical_list: C503463:20-methyl spirolide G; D019086:Bridged Bicyclo Compounds, Heterocyclic; D018733:Nicotinic Antagonists; D011725:Pyridines; D011978:Receptors, Nicotinic; D013141:Spiro Compounds; C082748:epibatidine
keywords: Xenopus oocytes; competition-binding assays; dinoflagellate toxin; molecular docking; neuromuscular transmission; nicotinic acetylcholine receptors; nicotinic currents; spirolides