AGONIST BINDING TO THE TORPEDO ACETYLCHOLINE-RECEPTOR .2. COMPLEXITIES REVEALED BY ASSOCIATION KINETICS

The binding of suberyldicholine to membrane-bound Torpedo acetylcholin e receptor has been monitored by fluorescence changes of covalently bo und 5-iodoacetamidosalicylic acid (IAS). At equilibrium, suberyldichol ine binds to two high-affinity binding sites (K-d approximate to 20 nM ). Kinetic experiments reveal that there is rapid formation of an init ial complex (K-d approximate to 2 mu M) which undergoes sequential fas t (k(app) approximate to 1 s(-1)) and slow (k(app) approximate to 0.05 s(-1)) conformational changes. These kinetics differ from those repor ted for other agonists [Blanchard, S. G., Dunn, S. M. J., & Raftery, M . A. (1982) Biochemistry 24, 6258-6264] in that, for suberyldicholine, there is no evidence for a second pathway involving the binding of an additional agonist molecule. These results, considered together with the observed dissociation kinetics (accompanying manuscript), suggest that each high-affinity site for acetylcholine is made up of two subsi tes, which suberyldicholine is able to bridge, thus occluding the bind ing of a second ligand. The kinetic mechanism for acetylcholine bindin g has been re-examined to accommodate the complexities of the [H-3]- a cetylcholine dissociation kinetics and the observation that, at equili brium, no more than two occupied binding sites are detected [accompany ing manuscript: Dunn, S. M. J., & Raftery, M. A. (1997) Biochemistry 3 6, 3846-3853]. It is suggested that, for each acetylcholine binding si te, a second ligand is able to bind but that the ternary complex is tr ansient since one of the two bound ligands again dissociates in the fo rmation of the equilibrium mono-liganded complex. To further probe the physical nature of the two subsites, the binding of a series of bis-q uaternary suberyldicholine analogues, (CH3)(3)N+CH2CH2OCO-(CH2)n-COOCH 2CH2N+(CH3)(3), to IAS-labeled receptor preparations has been examined . Analogues in which n <5 behave like acetylcholine, i.e., a second li gand binding pathway is observed, but longer ligands (n = 5-10) act li ke suberyldicholine and may be long enough to cross-link the sites.