Pharmacological and null mutation approaches reveal nicotinic receptor diversity

We have developed an array of assays for nicotinic acetylcholine receptor binding and function. [I-125]alpha-Bungarotoxin-, (-)-[H-3]nicotine-, and [H-3]epibatidine-binding nicotinic acetylcholine receptors were assayed in mouse brain membranes and sections. Nicotinic acetylcholine receptor function was quantified using synaptosomal [H-3]dopamine, [H-3]gamma-aminobutyric acid ([H-3]GABA), and Rb-86(+) efflux techniques. Additionally, the effects of beta 2 subunit deletion on each of the measures were assessed. Detailed pharmacological comparison revealed minimally six nicotinic binding subtypes: [I-125]alpha-bungarotoxin-binding nicotinic acetylcholine receptors; beta 2-subunit-dependent and -independent high-affinity(-)-[H-3]nicotine-binding sites; beta 2-dependent and -independent cytisine-resistant [H-3]epibatidine-binding sites; and a beta 2-dependent low-affinity [H-3]epibatidine binding site. Comparative pharmacology suggested that [H-3]GABA and dihydro-beta-erythroidine (DH beta E)-sensitive Rb-86(+) efflux are mediated by the same (probably alpha 4 beta 2) nicotinic acetylcholine receptor subtype, while other nicotinic acetylcholine receptor subtypes evoke [H-3]dopamine and DH beta E-resistant Rb-86(+) efflux. In whole-brain preparations, each measure of nicotinic acetylcholine receptor function was beta 2 dependent. The majority of beta 2-independent [H-3]epibatidine binding was located in small, scattered brain nuclei, suggesting that individual nuclei may prove suitable for identification of novel, native nicotinic acetylcholine receptors. (C) 2000 Elsevier Science B.V. All rights reserved.