Agonist binding to the Torpedo acetylcholine receptor. 2. Complexities revealed by association kinetics.

Abstract

The binding of suberyldicholine to membrane-bound Torpedo acetylcholine receptor has been monitored by fluorescence changes of covalently bound 5-iodoacetamidosalicylic acid (IAS). At equilibrium, suberyldicholine binds to two high-affinity binding sites (Kd approximately 20 nM). Kinetic experiments reveal that there is rapid formation of an initial complex (Kd approximately 2 microM) which undergoes sequential fast (k(app) approximately 1 s(-1)) and slow (k(app) approximately 0.05 s(-1)) conformational changes. These kinetics differ from those reported 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 subsites, which suberyldicholine is able to bridge, thus occluding the binding of a second ligand. The kinetic mechanism for acetylcholine binding has been re-examined to accommodate the complexities of the [3H]-acetylcholine dissociation kinetics and the observation that, at equilibrium, no more than two occupied binding sites are detected [accompanying manuscript: Dunn, S. M. J., & Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. It is suggested that, for each acetylcholine binding site, a second ligand is able to bind but that the ternary complex is transient since one of the two bound ligands again dissociates in the formation of the equilibrium mono-liganded complex. To further probe the physical nature of the two subsites, the binding of a series of bis-quaternary suberyldicholine analogues, (CH3)3N+CH2CH2OCO(CH2)n-COOCH2CH2N+(CH3)3, to IAS-labeled receptor preparations has been examined. Analogues in which n < 5 behave like acetylcholine, i.e., a second ligand binding pathway is observed, but longer ligands (n = 5-10) act like suberyldicholine and may be long enough to cross-link the sites.