Functional α6-containing nicotinic receptors are present in chick retina

Despite the fact that the neuronal chick alpha 6 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 alpha 6-containing nicotinic receptors in neuronal tissues. Using subunit-specific antibodies directed against two different epitopes of the chick alpha 6 subunit, we performed immunoprecipitation experiments on immunopurified alpha 6-containing receptors radiolabeled with the nicotinic agonist [H-3]epibatidine (Epi): almost all of the alpha 6 receptors contained the beta 4 subunit, 51% the beta 3 subunit, 42% the alpha 3 subunit, and 7.5% the beta 2 subunit. Western blot analyses of the purified receptors confirmed the presence of the alpha 3, beta 3, beta 2, and beta 4 subunits, and the absence of the alpha 4, alpha 5, and alpha 7 subunits. The alpha 6-containing receptors bind [H-3]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 alpha 6 receptors also have a distinct antagonist pharmacological profile with a rank order of potency of alpha-conotoxin MII > methyllycaconitine > dihydro-beta-erythroydine > MG624 > d-tubocurarine > decamethonium > hexamethonium. When reconstituted in lipid bilayers, the alpha 6-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.