[H-3]-methyllycaconitine: a high affinity radioligand that labels invertebrate nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) of insect and other invertebrates are heterogeneous and new tools are needed to dissect their multiplicity. [H-3]-Methyllycaconitine ([H-3]-MLA) is a novel radioligand which is a pote nt antagonist at vertebrate alpha7-type nAChR. Putative invertebrate nAChR of the aphid Myzus persicae, the moths Heliothis virescens and Manduca sext a, the fly Lucilia sericata, and the squid Loligo vulgaris were investigate d in radioligand binding studies with [H-3]-MLA. Saturable binding was cons istent with a single class of high affinity binding sites for each of these invertebrates, characterised by a dissociation constant, K-d, of approxima tely I nM and maximal binding capacities, B-max, between 749 and 1689 fmol/ mg protein for the insects and 14,111 fmol/mg protein for squid. [H-3]-MLA binding to M. persicae membranes was characterised in more detail. Kinetic analysis demonstrated rapid association in a biphasic manner and slow, mono phasic dissociation. Displacement studies demonstrate the nicotinic charact er of [H-3]-MLA binding sites. Data for all nicotinic ligands, except MLA i tself, are consistent with displacement from a high and a low affinity site , indicating that displacement is occurring from two or more classes of nic otinic binding site that are not distinguished by MLA itself. Autoradiograp hic analysis of the distribution of [H-3]-MLA binding sites in Manduca sext a shows discrete labelling of neuropil areas of the optic and antennal lobe s. (C) 2001 Elsevier Science Ltd. All rights reserved.