Marine Macrocyclic Imines, Pinnatoxins A and G: Structural Determinants and Functional Properties to Distinguish Neuronal α7 from Muscle α12βγδ nAChRs

Pinnatoxins are macrocyclic imine phycotoxins associated with algal blooms and shellfish toxicity. Functional analysis of pinnatoxin A and pinnatoxin G by binding and voltage-clamp electrophysiology on membrane-embedded neuronal alpha 7, alpha 4 beta 2, alpha 3 beta 2, and muscle-type alpha 1(2)beta gamma delta nicotinic acetylcholine receptors (nAChRs) reveals high-affinity binding and potent antagonism for the alpha 7 and alpha 1(2)beta gamma delta subtypes. The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP), with low K-d values reflecting slow dissociation. Crystal structures of pinnatoxin-AChBP complexes (1.9-2.2 angstrom resolution) show the multiple anchoring points of the hydrophobic portion, the cyclic imine, and the substituted bis-spiroketal and cyclohexene ring systems of the pinnatoxins that dictate tight binding between the opposing loops C and F at the receptor subunit interface, as observed for the 13-desmethyl-spirolide C and gymnodimine A congeners. Uniquely, however, the bulky bridged EF-ketal ring specific to the pinnatoxins extends radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity.