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.