Agonist Binding to the Torpedo Acetylcholine Receptor. 2. Complexities Revealed by Association Kinetics

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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 ≈ 20 nM). Kinetic experiments reveal that there is rapid formation of an initial complex (Kd ≈ 2 μM) which undergoes sequential fast (kapp ≈ 1 s-1) and slow (kapp ≈ 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) Biochemistry24, 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) Biochemistry36, 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 n = 5−10) act like suberyldicholine and may be long enough to cross-link the sites.