Abstract
Agonists turn on receptors because they have a higher affinity for active versus resting conformations of the protein. Activation can occur by either of two pathways that connect to form a cycle: Agonists bind to resting receptors that then become active, or resting receptors activate and then bind agonists. We used mutations to construct endplate acetylcholine receptors (AChRs) having only one functional neurotransmitter-binding site and single-channel electrophysiology to measure independently binding constants for four different agonists, to both resting and active conformations of each site. For all agonists and sites, the total free energy change in each pathway was the same, confirming the activation cycle without external energy. Other results show that (i) there is no cooperativity between sites; (ii) agonist association is slower than diffusion in resting receptors but nearly diffusional in active receptors; (iii) whereas resting affinity is determined mainly by agonist association, active affinity is determined mainly by agonist dissociation; and (iv) at each site and for all agonists, receptor activation approximately doubles the agonist-binding free energy. We discuss a two-step mechanism for binding that involves diffusion and a local conformational change ("catch") that is modulated by receptor activation. The results suggest that binding to a resting site and the switch to high affinity are both integral parts of a single allosteric transition. We hypothesize that catch ensures proper signal recognition in complex chemical environments and that binding site compaction is a determinant of both resting and active affinity.