Purification and characterization of an alpha-bungarotoxin receptor that forms a functional nicotinic channel.

Neither the structure nor the function of alpha-bungarotoxin (alpha Bgtx) binding molecules in the nervous system have yet been completely defined, although it is known that some of these molecules are related to cation channels and some are not. Using an improved method of affinity chromatography, we have isolated a toxin binding molecule from chicken optic lobe that contains at least three subunits with apparent Mr values of 52,000, 57,000, and 67,000. The Mr 57,000 subunit binds alpha Bgtx and seems to be present in two copies per receptor. The receptor is recognized by antibodies raised against the alpha Bgtx receptors of human neuroblastoma cells, fetal calf muscle, and chicken optic lobe but not by antibodies raised against Torpedo acetylcholine receptor, the serum of myasthenic patients, or monoclonal antibody, 35. 125I-labeled alpha Bgtx binding to the isolated receptor is blocked, with the same potency, by nicotinic agonists and antagonists, such as nicotine, neuronal bungarotoxin and, d-tubocurarine. When reconstituted in a planar lipid bilayer, the purified alpha Bgtx receptor forms cationic channels with a conductance of 50 pS. These channels are activated in a dose-dependent manner by carbamylcholine and blocked by d-tubocurarine.

The alpha-bungarotoxin receptor purified from a human neuroblastoma cell line: biochemical and immunological characterization.

The pharmacological and electrophysiological characteristics of the alpha-bungarotoxin receptor present on the human neuroblastoma cell line IMR-32 indicate that this receptor is not associated with an acetylcholine-operated ionic channel. In this paper we report its biochemical purification and immunological characterization. This molecule has a standard sedimentation coefficient of 10S and sodium dodecyl-sulphate gel electrophoresis shows that it is made up of three polypeptide chains of molecular weights of 67,000, 60,000 and 52,000. Ligand binding to blots of purified receptor revealed that only the polypeptide of molecular weight 52,000 is bound by [125I]alpha-bungarotoxin. The purified alpha-bungarotoxin receptor was bound by polyclonal antibodies raised against purified fetal calf, Torpedo and chick optic lobe nicotinic receptors and by the sera of myasthenic patients. Furthermore, despite the fact that a number of different immunological techniques were used, it was impossible to label this alpha-bungarotoxin receptor with mAb 35, a monoclonal antibody which binds some neuronal nicotinic receptors. Rabbit antisera against the purified alpha-bungarotoxin receptor were used to compare this protein with other known nicotinic receptors and, once again, it was demonstrated that there is some immunological cross-reactivity between the alpha-bungarotoxin receptor present on neuroblastoma cells and Torpedo, fetal calf and chick optic lobe nicotinic receptors. All these immunological data, together with previously published pharmacological and molecular biology data, demonstrate that the alpha-bungarotoxin receptor present in nerve cells is neither a muscular nor a neuronal nicotinic receptor, although it has similarities with both.