title: In vitro labelling of muscle type nicotinic receptors using a fluorophore-conjugated pinnatoxin F derivative.
abstract: Fluorescent molecules are regularly utilised to study ligand-receptor interactions. Many ligands for nicotinic receptors have been conjugated with fluorophores to study receptor kinetics, recycling and ligand binding characteristics. These include small agonist molecules, as well as large peptidic antagonists. However, no small molecule antagonists have been investigated using this method. Pinnatoxin F is a newly discovered non-peptidic muscle type nicotinic receptor antagonist produced by the marine dinoflagellate species Vulcanodinium rugosum. This molecule has the potential for conjugation to a fluorophore, allowing subsequent visualisation of interactions with nicotinic receptors. Pinnatoxin F was modified by addition of diaminopolyether spacers, to which a fluorophore (VivoTag(®) 645) was conjugated. The fluorescent pinnatoxin was then applied to muscle sections from thy1-YFP-H transgenic mice, which express YFP in motor nerves, to allow direct visualization of fluorescent binding at the neuromuscular junction. The addition of both the diaminopolyether spacer and the VivoTag(®) 645 reduced the potency of pinnatoxin F, as evidenced by a reduction in in vitro neuromuscular blocking activity and in vivo toxicity. Despite this reduced potency, the fluorescent molecule selectively labelled endplate regions in thy1-YFP mouse muscle sections and this labelling was inhibited by pre-exposure of muscle sections to native pinnatoxin F or the nicotinic antagonist α-bungarotoxin. This study proves nicotinic receptor binding activity of pinnatoxin F and is the first example of a fluorophore-conjugated small-molecule antagonist for nicotinic receptors. These results indicate the potential for other small-molecule nicotinic receptor antagonists to be fluorescently labelled and used as probes for specific nicotinic receptor subtypes.
authors.affiliation: Department of Pharmacology and Toxicology, University of Otago School of Medical Sciences, Dunedin, New Zealand.
authors.forename: Shane D
authors.initials: SD
authors.lastname: Hellyer
authors.affiliation: Cawthron Institute, 98 Halifax Street East, Private Bag 2, Nelson, New Zealand.
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Selwood
authors.affiliation: Cawthron Institute, 98 Halifax Street East, Private Bag 2, Nelson, New Zealand.
authors.forename: Roel
authors.initials: R
authors.lastname: van Ginkel
authors.affiliation: AgResearch, Private Bag 3123, Hamilton, New Zealand.
authors.forename: Rex
authors.initials: R
authors.lastname: Munday
authors.affiliation: Department of Physiology, University of Otago School of Medical Sciences, Dunedin, New Zealand.
authors.forename: Phil
authors.initials: P
authors.lastname: Sheard
authors.affiliation: National Veterinary Institute, PB 750 Sentrum, 0106 Oslo, Norway; Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, N-0316 Oslo, Norway.
authors.forename: Christopher O
authors.initials: CO
authors.lastname: Miles
authors.affiliation: Cawthron Institute, 98 Halifax Street East, Private Bag 2, Nelson, New Zealand.
authors.forename: Lesley
authors.initials: L
authors.lastname: Rhodes
authors.affiliation: Department of Pharmacology and Toxicology, University of Otago School of Medical Sciences, Dunedin, New Zealand. Electronic address: steve.kerr@stonebow.otago.ac.nz.
authors.forename: D Steven
authors.initials: DS
authors.lastname: Kerr
mesh_terms: D000470:Alkaloids / Q000138:chemical synthesis / Q000633:toxicity; D000818:Animals; D005456:Fluorescent Dyes; D007928:Lethal Dose 50; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009469:Neuromuscular Junction; D018733:Nicotinic Antagonists / Q000138:chemical synthesis / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000187:drug effects*; D012132:Respiratory Muscles / Q000187:drug effects / Q000378:metabolism*; D013141:Spiro Compounds / Q000138:chemical synthesis / Q000633:toxicity
chemical_list: D000470:Alkaloids; D005456:Fluorescent Dyes; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D013141:Spiro Compounds; C552839:pinnatoxin F
keywords: Confocal imaging; Endplate; Fluorescent pinnatoxin; NMJ; Nicotinic receptors