Characterisation of the binding of [H-3]methyllycaconitine: a new radioligand for labelling alpha 7-type neuronal nicotinic acetylcholine receptors

Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphiniu m seeds, is one of the most potent non-proteinacious ligands that is select ive for abungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [H-3]MLA bound to rat brain membranes with high affinity (K-d = 1. 86 +/- 0.31 nM) with a good ratio of specific to non-specific binding. The binding of [H-3]MLA was characterised by rapid association (t(1/2) = 2.3 mi n) and dissociation (t(1/2) = 12.6 min) kinetics. The radioligand binding d isplayed nicotinic pharmacology, consistent with an interaction with alpha bungarotoxin-sensitive nAChR. The snake alpha-toxins, alpha bungarotoxin an d alpha cobratoxin, displaced [H-3]MLA with high affinity (K-i = 1.8 +/- 0. 5 and 5.5 +/- 0.9 nM, respectively), whereas nicotine was less potent (K-i = 6.1 +/- 1.1 mu M). The distribution of [H-3]MLA binding sites in crudely dissected rat brain regions was identical to that of [I-125]alpha bungaroto xin binding sites, with a high binding site density in hippocampus and hypo thalamus, but low density in striatum and cerebellum. [H-3]MLA also labelle d a sub-population of binding sites which are not sensitive to the snake al pha toxins, but which did not differ significantly from the major populatio n with respect to their other pharmacological properties or regional distri bution. [H-3]MLA, therefore, is a novel radiolabel for characterising alpha 7-type nAChR. A good signal to noise ratio and rapid binding kinetics prov ide advantages over the use of radiolabelled alpha bungarotoxin for rapid a nd accurate equilibrium binding assays. (C) 1999 Elsevier Science Ltd. All rights reserved.