Abstract

Despite the fact that the neuronal chick α6 subunit was first cloned several years ago and recently has been shown to form acetylcholine (ACh)-activated channels in heterologous systems, no information is yet available concerning the structure and function of the α6-containing nicotinic receptors in neuronal tissues. Using subunit-specific antibodies directed against two different epitopes of the chick α6 subunit, we performed immunoprecipitation experiments on immunopurified α6-containing receptors radiolabeled with the nicotinic agonist [³H]epibatidine (Epi): almost all of the α6 receptors contained the β4 subunit, 51% the β3 subunit, 42% the α3 subunit, and 7.5% the β2 subunit. Western blot analyses of the purified receptors confirmed the presence of the α3, β3, β2, and β4 subunits, and the absence of the α4, α5, and α7 subunits. The α6-containing receptors bind [³H]Epi (K _d = 35 pM) and a number of other nicotinic agonists with very high affinity, the rank order being Epi ≫ cytisine > nicotine > 1,1-dimethyl-4-phenylpiperazinium > acetylcholine > carbamylcholine. The α6 receptors also have a distinct antagonist pharmacological profile with a rank order of potency of α-conotoxin MII > methyllycaconitine > dihydro-β-erythroydine > MG624 > d-tubocurarine > decamethonium > hexamethonium. When reconstituted in lipid bilayers, the α6-containing receptors form functional cationic channels with a main conductance state of 48 pS. These channels are activated by nicotinic agonists in a dose-dependent manner, and blocked by the nicotinic antagonist d-tubocurarine.