Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at alpha delta and either alpha epsilon (adult) or alpha gamma (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (Delta G(B1)). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline,Delta G(B1) is similar to-2 kcal/mol more favorable at alpha gamma compared with at alpha epsilon and alpha delta. Only three of the aromatics contribute significantly to Delta G(B1) at the adult sites (alpha Y190, alpha Y198, and alpha W149), but all five do so at alpha gamma (as well as alpha Y93 and gamma W55). gamma W55 makes a particularly large contribution only at alpha gamma that is coupled energetically to those contributions of some of the a-subunit aromatics. The hydroxyl and benzene groups of loop C residues alpha Y190 and alpha Y198 behave similarly with regard to Delta G(B1) at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the alpha gamma site is more compact, better organized, and less dynamic than alpha epsilon and alpha delta. We speculate that the different sensitivities of the fetal alpha gamma site versus the adult alpha epsilon and alpha delta sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse.