Functional alpha 6-containing nicotinic receptors are present in chick retina

Despite the fact that the neuronal chick alpha 6 subunit was first cloned s everal years ago and recently has been shown to form acetylcholine (ACh)-ac tivated channels in heterologous systems, no information is yet available c oncerning the structure and function of the alpha 6-containing nicotinic re ceptors in neuronal tissues. Using subunit-specific antibodies directed aga inst two different epitopes of the chick alpha 6 subunit, we performed immu noprecipitation 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 subun it, 42% the alpha 3 subunit, and 7.5% the beta 2 subunit. Western blot anal yses 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, an d alpha 7 subunits. The alpha 6-containing receptors bind [H-3]Epi (K-d = 3 5 pM) and a number of other nicotinic agonists with very high affinity, the rank order being Epi >> cytisine > nicotine > 1,1-dimethyl-4-phenylpiperaz inium > 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 > MG6 24 > d-tubocurarine > decamethonium > hexamethonium. When reconstituted in lipid bilayers, the alpha 6-containing receptors form functional cationic c hannels with a main conductance state of 48 pS. These channels are activate d by nicotinic agonists in a dose-dependent manner, and blocked by the nico tinic antagonist d-tubocurarine.