Chemical modification of the nicotinic cholinergic receptor of PC-12 nerve cell.

The identity of the protein that mediates the nicotinic acetylcholine sensitivity in neuronal cells has been investigated by chemical modification and affinity labeling. When an ion flux assay is used, it is possible to measure specifically the activity of the ionophore associated with the nicotinic acetylcholine receptor in cultured nerve cells (PC-12 pheochromocytoma). This activity is modulated by modification of the redox state of at least one disulfide bridge located at the vicinity of the agonist binding site. The oxidizing agent 5,5'-dithiobis(nitrobenzoic acid) restores the complete receptor response which had been inhibited by reduction with dithiothreitol. N-Ethylmaleimide and the nicotinic affinity labels [4-(N-maleimido)benzyl]-alpha-trimethylammonium iodide and bromoacetylcholine react also with the reduced receptor and irreversibly block the agonist-dependent response of the ionophore. The two affinity labels show strong affinities for the receptor, and apparent IC50 values of 20 and 560 nM can be respectively evaluated. Bromoacetylcholine, being an acetylcholine analogue, blocks the receptor function by desensitization, a process in which the constant interaction with the activator causes a shift into an inactive form of the receptor. Bromoacetylcholine can also be shown to activate untreated as well as reduced cells. In this case, the bound label induces a lasting response which is terminated by the irreversible desensitization of the modified receptor. These experiments thus show that the PC-12 nicotinic ionophore shares functional and structural similarities with peripheral receptors. They suggest that nicotinic affinity labels developed for the muscle receptor can also be used as specific markers of the nicotinic neural ionophore.