Molecular recognition at cholinergic synapses: acetylcholine versus choline

Abstract

Key points: Neuromuscular acetylcholine (ACh) receptors have a high affinity for the neurotransmitter ACh and a low affinity for its metabolic product choline. At each transmitter binding site three aromatic groups determine affinity, and together provide ∼50% more binding energy for ACh than for choline. Deprotonation of αY190 by a nearby lysine strengthens the interaction between this aromatic ring and both ACh and choline. H-bonds position ACh and choline differently in the aromatic cage to generate the different affinities.

Abstract: 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 αY190, αW149 and αY198 each provide ∼50% less binding energy for Cho compared to ACh. At αY198 a phenylalanine substitution had no effect, but at αY190 this substitution caused a large, agonist-independent loss in binding energy that depended on the presence of αK145. The results suggest that (1) αY190 is deprotonated by αK145 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.

Keywords: agonist; cation-pi; ion channel; neuromuscular; receptor.

Figure 1. AChR and the aromatic cage

A, low‐resolution view of the Torpedo AChR (PDB accession number 2BG9). Each transmitter binding site is at a subunit interface (box). B, close‐up of the ligand binding site of an acetylcholine binding protein (AChBP; PDB accession number 1UV6). Three α‐subunit aromatics (green) form a cage around carbamylcholine (CCh, white); these determine affinity in adult AChRs but in fetal‐type αY93 and γW55 (yellow) also make a contribution. Black dashed line, interaction between αK145 and αY190. Numbers are mouse AChRs. C, close‐up of the α‐subunit aromatic cage. Top, X‐ray structure of AChBP bound with ACh (PDB accession number 3WIP). Dashed line, H‐bond to a structural water (red sphere). Bottom, homology model of Cho in the mouse α–δ binding site. Dashed line, H‐bond to the αW149 backbone carbonyl. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. Single‐channel currents activated by Cho or TMA

A, at low time resolution, αW149A openings are clustered (100 mm Cho, +70 mV, openings are down). Within a cluster a single AChR oscillates between C(losed) and O(pen), and between clusters all AChRs in the patch are D(esensitized). B, higher time resolution views of clusters. The background was either WT or ε(L269F+E181W), which increased the unliganded gating equilibrium constant 1492‐fold. The binding site mutations all reduce P_O.

Figure 3. Effects of mutations on binding free energy

A, all mutations result in a positive binding free energy change (decrease affinity) for all agonists. αY190A has the largest effect. The αK145A mutation has no effect when combined with αY190F. B, graphical summary. Numbers are free energy losses (kcal mol⁻¹) resulting from the removal of an OH (Y‐to‐F) or a ring (W/F‐to‐A). For all agonists, indole>benzene; for all rings, ACh>Cho. For clarify, only the agonist's quaternary nitrogen is visualized as a blue sphere. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Effects of OH removal at αY190 and αY198 are agonist‐independent

Removal of the hydroxyl group of αY190 causes a ∼+2.0 kcal mol⁻¹ reduction in binding energy for six different agonists, but removal of a hydroxyl group of αY198 has little effect.

Figure 5. Single‐site analyses

The change in binding energy with αW149A and αY198A is approximately equal at both adult‐type binding sites.