Activation of endplate nicotinic acetylcholine receptors by agonists

Abstract

The interaction of a small molecule made in one cell with a large receptor made in another is the signature event of cell signaling. Understanding the structure and energy changes associated with agonist activation is important for engineering drugs, receptors and synapses. The nicotinic acetylcholine receptor (AChR) is a ∼300kD ion channel that binds the neurotransmitter acetylcholine (ACh) and other cholinergic agonists to elicit electrical responses in the central and peripheral nervous systems. This mini-review is in two sections. First, general concepts of skeletal muscle AChR operation are discussed in terms of energy landscapes for conformational change. Second, adult vs. fetal AChRs are compared with regard to interaction energies between ACh and agonist-site side chains, measured by single-channel electrophysiology and molecular dynamics simulations. The five aromatic residues that form the core of each agonist binding site can be divided into two working groups, a triad (led by αY190) that behaves similarly at all sites and a coupled pair (led by γW55) that has a large influence on affinity only in fetal AChRs. Each endplate AChR has 5 homologous subunits, two of α(1) and one each of β, δ, and either γ (fetal) or ϵ (adult). These nicotinic AChRs have only 2 functional agonist binding sites located in the extracellular domain, at αδ and either αγ or αϵ subunit interfaces. The receptor undergoes a reversible, global isomerization between structures called C and O. The C shape does not conduct ions and has a relatively low affinity for ACh, whereas O conducts cations and has a higher affinity. When both agonist sites are empty (filled only with water) the probability of taking on the O conformation (PO) is low, <10(-6). When ACh molecules occupy the agonist sites the C→O opening rate constant and C↔O gating equilibrium constant increase dramatically. Following a pulse of ACh at the nerve-muscle synapse, the endplate current rises rapidly to reach a peak that corresponds to PO ∼0.96.

Keywords: Acetylcholine; Allosteric; Binding; Gating; Neuromuscular; Nicotinic.

Figure 1. Energy landscape for adult endplate AChRs

Without agonists, C(losed)-to-O(pen) gating is uphill (+8.3 kcal/mol; gray arrow). This energy change becomes downhill with 2 ACh molecules bound to the agonist sites, because the affinity of O is higher than that of C (−10.2 kcal/mol; the difference between the green arrows). Red arrow, C↔O oscillations that give rise to clusters of single-channel openings (see Fig. 2).

Figure 2. Clusters

A. Activation by agonists. Left, macroscopic current (outside-out patch). A sustained step of 1 mM ACh causes a rapid increase in P_O followed by a sag, as receptors enter D states [28]. Right, single-channel currents (cell-attached patch; O is down). Clusters are C↔O oscillations and the long, silent periods between clusters are sojourns in D. Aligning and averaging clusters will reproduce the macroscopic current. Bottom, close up of a cluster (30 μM ACh, adult-type mouse endplate AChRs expressed in HEK cells, 23 °C, −100 mV). B. Activation by mutations (no agonists). Top, WT endplate AChRs rarely open without agonists. Bottom, adding background mutations that increase the allosteric constant (make the gray arrow more negative, in Fig. 1) but have no effect on binding generates clusters (αY127F+αD97A+αP272A) [29]. Like agonists, these mutations ‘tilt’ the energy landscape downward, but without changing affinities (green arrows in Fig. 1).

Figure 3. The Core

The principal side of the binding pocket (α, in AChRs) is white and the complementary side (δ, ε or γ) is tan. Aromatic triad residues are green and special pair residues are yellow. Structure is AChBP (pdb accession number 1uv6 [30]). Agonist is carbamylcholine (CCh); numbering is for mouse endplate AChRs.

Figure 4. Breaking down binding energy at the Core

The area of each slice is approximately equal to the loss in favorable ACh binding free energy with the removal of just that group. Dashed gray lines separate −OH and ring contributions of tyrosines. The special pair makes a larger contribution at the fetal αγ site, whereas the action of the aromatic triad is similar at all sites (αε is like αδ).

Figure 5. MD simulations

a. In silico and in vivo ACh binding energies match. The histogram entries are energies calculated from the MD trajectories and the white circles are the corresponding free energies estimated from single-channel currents. b. Representative snapshots from the MD trajectories. The different positions of aromatic triad and special pair residues in αγ (green) vs. αδ (gray) are consistent with the in vivo energy differences (Fig. 4). Blue ball, quaternary ammonium (QA) group of ACh.