Single-channel fluctuations of a chloride-specific channel from Torpedo californica electroplax were studied with high current and time resolution . Channels were incorporated into virtually solvent-free planar bilayer membranes formed from phospholipid monolayers, and the substructure of the open channel was analyzed . The single channel displays three well-defined substates of conductances 0, 10, and 20 pS in 200 mM Cl -. These three substates are interpreted in terms of a dimeric channel complex composed of two identical "protochannels" gating independently in parallel on a time scale of milliseconds, but coupled together by a bursting process on a time scale of seconds. The probability of forming an open protochannel is voltage dependent and is increased strongly as aqueous pH is lowered. Variations of pH are effective only on the same side of the bilayer as the addition of electroplax vesicles . The dependence of singlechannel kinetics on pH and voltage lead to a minimal four-state model in which both open and closed states can be protonated on a residue that changes its pK from 6 to 9 upon opening of the protochannel .

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Despite the fact that the neuronal chick alpha6 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 alpha6-containing nicotinic receptors in neuronal tissues. Using subunit-specific antibodies directed against two different epitopes of the chick alpha6 subunit, we performed immunoprecipitation experiments on immunopurified alpha6-containing receptors radiolabeled with the nicotinic agonist [3H]epibatidine (Epi): almost all of the alpha6 receptors contained the beta4 subunit, 51% the beta3 subunit, 42% the alpha3 subunit, and 7.5% the beta2 subunit. Western blot analyses of the purified receptors confirmed the presence of the alpha3, beta3, beta2, and beta4 subunits, and the absence of the alpha4, alpha5, and alpha7 subunits. The alpha6-containing receptors bind [3H]Epi (Kd = 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 alpha6 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 alpha6-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.