CHOLINERGIC BINDING-SITES ON THE PENTAMERIC ACETYLCHOLINE-RECEPTOR OFTORPEDO-CALIFORNICA

The binding of agonists, antagonists, and the acetylcholinesterase inh ibitor, eserine, to the nicotinic acetylcholine receptor from Torpedo californica has been monitored by the fluorescence changes of two extr insic probes that have been covalently attached to the receptor protei n. Although both probes, 5-(iodoacetamido)salicylic acid (IAS) and thy l]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD) react with sul fhydryl groups, they do not react at the same location. The conditions for IAS labeling and competition studies have shown that, following r eduction of the receptor, this fluorophore reacts with the same cystei nes on each of the two alpha subunits that may be labeled by the alkyl ating agonist, [H-3]bromoacetylcholine. The fluorescence of this probe is sensitive to the binding of agonists and competitive antagonists t o two high-affinity sites on the receptor. IANBD does not react with t he same cysteines as IAS, and its fluorescence is unchanged by the hig h-affinity binding of agonists and antagonists. The fluorescence of th is probe is, however, specifically and saturably enhanced by the bindi ng of agonists to distinct low-affinity sites. Heterogeneity in the NB D fluorescence changes induced by the bis-quaternary agonist, suberyld icholine, has indicated that the stoichiometry of low-affinity sites i s also two per receptor. Stopped-flow studies of agonist binding to re ceptor preparations that had been doubly labeled by both fluorophores demonstrate that the conformational changes detected by IAS occur on s low time scales of seconds to minutes whereas a much faster conformati onal change is revealed by changes in NBD fluorescence. These results provide further evidence that the acetylcholine receptor carries two d istinct classes of sites for classical agonists and are consistent wit h the involvement of the low-affinity sites in channel activation. Ese rine, which has been reported to activate the acetylcholine receptor c hannel at a concentration of 50-100 muM via a pathway that is not inhi bited by alpha-bungarotoxin [Okonjo, K. O., Kuhlmann, J., & Maelicke, A. (1991) Eur. J. Biochem. 200, 671-677], also induces a saturable enh ancement of NBD fluorescence (K(d) approximately 50 muM) that is not b locked by alpha-bungarotoxin. These results are consistent with the no tion that the NBD fluorescence changes induced by high concentrations of agonists are a reflection of the induction of cation translocation.