Role of surface negative charges in agonist binding to the unliganded open state of the neuromuscular acetylcholine receptor

Acetylcholine receptors (AChRs) expressed at the nerve muscle junction (NMJ) synapses are hetero-pentameric ligand-gated ion channels with two neurotransmitter binding sites (TBS) at -{delta} and -{varepsilon} (adult)/{gamma} (fetal) subunit interfaces. They are typical allosteric proteins which reside in at least two stable conformations: resting (Closed) and active (Open) states. Mechanism of agonist (A) binding to the C state has been extensively studied, but agonist association to the O state has not been clearly understood. Here, by using engineered constitutively active AChRs, single-channel patch-clamp, and molecular dynamics (MD) simulations, we elucidate the differences between unliganded (O) vs liganded (AO) active states and the mechanism of agonist association to the O state. Our results indicate that: 1. At the {gamma}-binding site, for a series of agonists, the O state agonist association rate (jon) was [~]30 times faster than the diffusion limit and agonist affinity was 2-orders of magnitude higher vs at the {delta}- and {varepsilon}-binding sites. 2. Electrostatic surface charge density owing to 4 negatively charged residues ({gamma}E57, {gamma}D113, {gamma}D174, and {gamma}E180) facilitates loop C capping, compaction of the TBS, and agonist stabilization in the AO state. 3. Mutating these residues in combination reversed the jon and binding affinity to those comparable to the {delta}- and {varepsilon}-binding sites. 4. {varphi}-value analysis indicated the presence of a transition state intermediate between the O and AO states. Overall, we elucidate the role of neighboring charged residues outside the TBS in determining high-affinity agonist binding and their significance in shaping the synaptic response at the NMJ. SummaryNicotinic acetylcholine receptors (AChRs) are allosteric proteins that are crucial for muscle contraction. The isomerization of resting closed (C) to active open (O) state can be achieved by both bind-gate and gate-bind (rare) pathway. Little is known about agonist binding to the unliganded functional O state. Here, we elucidate the nature of unliganded O state and the mechanism of agonist binding to this high-affinity O state by single-channel current recordings, protein engineering, in silico methods and phi-analysis. Our results describe an energetic barrier between the unliganded and liganded O state owing to negatively charged amino acids near the agonist site. These charges partially contribute to the low- and high-affinity agonist energies specifically at the embryonic neurotransmitter binding site. We also discussed the physiological significance of these residues in shaping synaptic response. Significance StatementEmbryonic-type AChRs are indispensable for nerve-muscle junction formation. Many mutations in these receptors cause congenital myasthenia syndrome by increasing constitutive channel activation. Therefore, understanding the nature of unliganded functional open (O) state is critical. Here, we describe the nature and the mechanism of agonist binding to the unliganded O (or apo) state. Single-channel currents were recorded with different background constructs and binding site mutations. The results highlight the importance of loop C and ligand orientation in determining high-affinity agonist binding. Further we elucidate the role of electrostatic interactions in agonist binding to the O state (high-affinity binding). We surmise that higher association rate of agonist binding to O state is the reason behind higher efficacy of the fetal receptor.