Acetylcholine receptor channels activated by a single agonist molecule

Abstract

The neuromuscular acetylcholine receptor (AChR) is an allosteric protein that alternatively adopts inactive versus active conformations (R<-->R). The R shape has a higher agonist affinity and ionic conductance than R. To understand how agonists trigger this gating isomerization, we examined single-channel currents from adult mouse muscle AChRs that isomerize normally without agonists but have only a single site able to use agonist binding energy to motivate gating. We estimated the monoliganded gating equilibrium constant E(1) and the energy change associated with the R versus R change in affinity for agonists. AChRs with only one operational binding site gave rise to a single population of currents, indicating that the two transmitter binding sites have approximately the same affinity for the transmitter ACh. The results indicated that E(1) approximately 4.3 x 10(-3) with ACh, and approximately 1.7 x 10(-4) with the partial-agonist choline. From these values and the diliganded gating equilibrium constants, we estimate that the unliganded AChR gating constant is E(0) approximately 6.5 x 10(-7). Gating changes the stability of the ligand-protein complex by approximately 5.2 kcal/mol for ACh and approximately 3.3 kcal/mol for choline.

Figure 1

αW149 mutations allow spontaneous gating but eliminate gating by 500 μM ACh. (A) Spontaneous activity (in the absence of agonists) of AChRs with three gain-of-function background mutations that greatly increase the unliganded gating equilibrium constant, E₀ (αD97A+αY127F+αS269I). Top: Single-channel currents from the background construct alone. Each cluster represents the R↔R^∗ activity of a single, unliganded AChR (conducting is down); long gaps between clusters are periods when all AChRs in the patch are desensitized; bottom trace is expanded view of marked currents. Middle and bottom: Spontaneous currents after adding a binding-site (αW149) mutation (Met or Leu, respectively). These mutations have only a modest effect on E₀ (Table 1). (B) Activity of AChRs exposed to 500 μM ACh (wt background, which shows very little spontaneous gating). Top: The open probability within clusters is high because the AChRs without any αW149 mutations are able to bind and use energy from ACh at both transmitter binding sites. Middle and bottom: The binding-site (αW149) mutations reduce the frequency of openings because they are unable to bind ACh or use energy from the agonist affinity change to trigger gating. Left and right calibrations are for low- and high-resolution views throughout.

Figure 2

Gating activity of monoliganded (hybrid) AChRs activated by ACh. (A) Activity of hybrid AChRs (wt background) exposed to 500 μM ACh. The AChRs had a combination of αW149 and αW149M residues at the two transmitter binding sites. The bottom two traces are marked openings at an expanded scale. Diliganded AChRs with two αW149 residues (wt; W-W) can use ACh binding energy to open efficiently and therefore produce high open-probability clusters. Monoliganded (hybrid) AChRs with one αW149 and one αW149M (hybrid; W-M and M-W) can use ACh binding energy at only one transmitter binding site and therefore produce lower open-probability clusters. (B) Activity of fully liganded, hybrid AChRs with the gain-of-function background mutation αS269I (Table 1). The AChRs had a combination of αW149 and αW149M residues at the two transmitter binding sites. In the low-resolution view, the sporadic, isolated openings reflect M-M AChRs that cannot utilize ACh binding energy. Marked clusters from wt and hybrid AChRs are shown below. (C) Cluster population analysis (αS269I background). Left: Both diliganded (W-W) high open-probability clusters (0.93; solid circles) and monoliganded (W-M and M-W) low open-probability clusters (0.19; open circles) are apparent in a single patch. Right: The monoliganded population, fitted by a single Gaussian. There is only one monoliganded population. (D) The conducting and nonconducting intervals within the monoliganded clusters are each fitted by a single exponential.

Figure 3

ACh binding parameters for monoliganded AChRs. (A) Results for αW149M hybrid AChRs (background, αS269I) activated by various [ACh]. For each panel: (left) example hybrid clusters; (right) intracluster, nonconducting interval histograms and probability density functions (solid line) computed from global model-fitting across concentrations (see Materials and Methods). The estimated rate constants (±SD) were as follows: association 153 ± 53 μM⁻¹s⁻¹ and dissociation 19,969 ± 9410 s⁻¹ (equilibrium dissociation constant: 131 μM; number of intervals: 5198). The published estimates for the association and dissociation rate constants for wt AChRs, assuming equal binding sites, are 167 μM⁻¹ s⁻¹ and 24,745 s⁻¹, respectively (18). (B) Results for αW149L hybrid AChRs (background, αS269I) activated by various [ACh]. The estimated rate constants were as follows: association, 126 ± 32 μM⁻¹s⁻¹; dissociation, 18,099 ± 4609 s⁻¹ (equilibrium dissociation constant: 144 μM; number of intervals: 10,218).

Figure 4

Gating activity of monoliganded (hybrid) AChRs activated by choline. (A) Activity of fully liganded and hybrid AChRs with the gain-of-function background mutation αP272A and activated by choline (Table 1). The AChRs had a combination of αW149 and αW149M residues at the two transmitter binding sites. Marked clusters from wt and hybrid AChRs are shown below. (B) Cluster population analysis. Left: Both diliganded (W-W) high open-probability clusters (0.80; solid circles) and monoliganded (W-M and M-W) low open-probability clusters (0.07; open circles) are apparent in a single patch. Right: The monoliganded population, fitted by a single Gaussian. There is only one population of monoliganded AChRs.