Pharmacological and null mutation approaches reveal nicotinic receptor diversity

Abstract

We have developed an array of assays for nicotinic acetylcholine receptor binding and function. [ 125I]α-Bungarotoxin-, (−)-[ 3H]nicotine-, and [ 3H]epibatidine-binding nicotinic acetylcholine receptors were assayed in mouse brain membranes and sections. Nicotinic acetylcholine receptor function was quantified using synaptosomal [ 3H]dopamine, [ 3H]γ-aminobutyric acid ([ 3H]GABA), and 86Rb + efflux techniques. Additionally, the effects of β2 subunit deletion on each of the measures were assessed. Detailed pharmacological comparison revealed minimally six nicotinic binding subtypes: [ 125I]α-bungarotoxin-binding nicotinic acetylcholine receptors; β2-subunit-dependent and -independent high-affinity (−)-[ 3H]nicotine-binding sites; β2-dependent and -independent cytisine-resistant [ 3H]epibatidine-binding sites; and a β2-dependent low-affinity [ 3H]epibatidine binding site. Comparative pharmacology suggested that [ 3H]GABA and dihydro-β-erythroidine (DHβE)-sensitive 86Rb + efflux are mediated by the same (probably α4β2) nicotinic acetylcholine receptor subtype, while other nicotinic acetylcholine receptor subtypes evoke [ 3H]dopamine and DHβE-resistant 86Rb + efflux. In whole-brain preparations, each measure of nicotinic acetylcholine receptor function was β2 dependent. The majority of β2-independent [ 3H]epibatidine binding was located in small, scattered brain nuclei, suggesting that individual nuclei may prove suitable for identification of novel, native nicotinic acetylcholine receptors.