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 αδ and either αε (adult) or αγ (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 (ΔG_B1). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔG_B1 is ∼−2 kcal/mol more favorable at αγ compared with at αε and αδ. Only three of the aromatics contribute significantly to ΔG_B1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔG_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 αγ site is more compact, better organized, and less dynamic than αε and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αε and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse.Receptors at synapses respond to specific chemical signals in the extracellular environment because the active conformation of the protein has a higher affinity for the ligand compared with the resting conformation (1, 2). The active vs. resting difference in binding free energy increases the relative stability of the active state and, hence, the probability of a cellular response. In this report, we describe and distinguish sources of ligand-binding free energy in three kinds of agonist site present in mouse muscle nicotinic acetylcholine receptors (AChRs). Our goal was to use single-channel electrophysiology to assess the relative contribution of significant functional groups to the overall free energy generated by the affinity change at each type of site.At cholinergic synapses, the main chemical signals are ACh released from nerve terminals and choline, which is an ACh precursor, hydrolysis product, and stable component of serum (3). The muscle AChR has central pore surrounded by five subunits of composition α₂βδε in adult-type and α₂βδγ in fetal-type (Fig. 1A) (4). The fetal, γ, subunit is essential for proper synapse maturation, and the adult, ε, subunit is necessary for proper function of mature synapses (5–7). Each AChR pentamer has two agonist binding sites in the extracellular domain, at αδ and either αε (adult) or αγ (fetal) subunit interfaces.Open in a separate windowFig. 1.Ligand binding sites. (A) Side view of a muscle AChR [Torpedo marmorata; PDB ID code 2bg9 (34)] showing an agonist site in the extracellular domain (αW149 and loops A, B, and C are marked). (Inset) Each AChR has two sites (filled circles) at αδ and αε (adult) or αγ (fetal) subunit interfaces. (B) High-resolution view of the ligand binding site of an acetylcholine binding protein occupied by carbamylcholine (CCh) [Lymnaea stagnalis; PDB ID code 1uv6 (11)]. Aromatic residues are labeled using mouse AChR numbering.The change in agonist affinity occurs within the global, resting↔active “gating” conformational change. Structural rearrangements at agonist sites that generate the affinity change are akin to movements of S4 in voltage-gated channels that generate gating currents. Given the central role of receptors at synapses, we thought it important to understand in detail the components of the free energy change that undergird the agonist affinity change. In wild-type AChRs, a large, uphill gating energy without agonists ensures the system will rarely activate constitutively, and a large, downhill free energy generated by affinity increases at the two agonist sites ensures that the protein will be active with a high probability after the release of ACh from the motor nerve terminal (8).We have estimated the free energy contributions of eight functional groups of five conserved residues at three different kinds of muscle AChR agonist site (αδ, αε, and αγ). On the α side of each site, there are four aromatics known to influence agonist affinity: αY190 (in loop C), αY198 (loop C), αY93 (loop A), and αW149 (loop B) (Fig. 1) (9–13). In addition, there is a conserved tryptophan in the nonα subunit, W55 (at position 57 in the δ subunit) (11, 14–16). In fetal AChRs, αW149 and αY198 have been shown to stabilize the quaternary ammonium of the agonist by cation-π forces (10, 13, 17).Previously, estimates of the ACh-binding free energy difference in mouse adult-type receptors after mutations indicated that only three of the mentioned aromatics (αY190, αY198, and αW149) are important (18), and other experiments showed that the free energy difference from both agonist sites combined is greater in fetal vs. adult AChRs (19). Here, we extend and refine these estimates. First, we measured the change in the net binding free energy after a mutation of each aromatic side chain in AChRs having just one functional binding site, so that the αδ, αε, and αγ sites could be probed independently, rather than pairwise. Second, we made some of these measurements using three partial agonists in addition to ACh, including the physiological ligand choline. Third, we estimated the degree of free energy coupling between some of the aromatic side chains at the fetal, αγ, site. Fourth, we used molecular dynamics (MD) simulations to estimate ACh binding energies and suggest structural correlates for differences between the three types of agonist site. We hypothesize that a greater sensitivity of fetal vs. adult AChRs to choline is a reason for the γ→ε subunit swap required for proper maturation of the neuromuscular synapse.