Molecular recognition at cholinergic synapses: acetylcholine versus choline

Acetylcholine (ACh) released at the vertebrate nerve-muscle synapse is hydrolysed rapidly to choline (Cho), so endplate receptors (AChRs) are exposed to high concentrations of both of these structurally related ligands. To understand how these receptors distinguish ACh and Cho, we used single-channel electrophysiology to measure resting affinities (binding free energies) of these and other agonists in adult-type mouse AChRs having a mutation(s) at the transmitter-binding sites. The aromatic rings of Y190, W149 and Y198 each provide approximate to 50% less binding energy for Cho compared to ACh. At Y198 a phenylalanine substitution had no effect, but at Y190 this substitution caused a large, agonist-independent loss in binding energy that depended on the presence of K145. The results suggest that (1) Y190 is deprotonated by K145 to strengthen the interaction between this benzene ring and the agonist's quaternary ammonium (QA) and (2) AChRs respond strongly to ACh because an H-bond positions the QA to interact optimally with the rings, and weakly to Cho because a different H-bond tethers the ligand to misalign the QA and form weaker interactions with the aromatic groups. The results suggest that the difference in ACh versus Cho binding energies is determined by different ligand positions within a fixed protein structure.