Thinking in cycles: MWC is a good model for acetylcholine receptor-channels

Abstract

Neuromuscular acetylcholine receptors have long been a model system for understanding the mechanisms of operation of ligand-gated ion channels and fast chemical synapses. These five subunit membrane proteins have two allosteric (transmitter) binding sites and a distant ion channel domain. Occupation of the binding sites by agonist molecules transiently increases the probability that the channel is ion-permeable. Recent experiments show that the Monod, Wyman and Changeux formalism for allosteric proteins, originally developed for haemoglobin, is an excellent model for acetylcholine receptors. By using mutations and single-channel electrophysiology, the gating equilibrium constants for receptors with zero, one or two bound agonist molecules, and the agonist association and dissociation rate constants from both the closed- and open-channel conformations, have been estimated experimentally. The change in affinity for each transmitter molecule between closed and open conformations provides ~-5.1 kcal mol(-1) towards the global gating isomerization of the protein.

Figure 1. The MWC model for AChRs

R is the low-affinity, closed-channel conformation, R* is the high-affinity, open-channel conformation and A is the agonist. K_d and J_d are the agonist equilibrium dissociation constants to R and R*. E_n is the gating equilibrium constant with n bound agonist molecules. The two binding sites of the adult mouse AChR are equivalent. Desensitized states (not shown) would be attached to both R and R* states, although this process mainly proceeds from R*. The boxed states are the ‘main’ states of the AChR that generate cellular responses. The unboxed states are rarely visited but knowledge of the corresponding equilibrium constants is essential because they complete the cycle and, therefore, allow the estimation of λ=K_d/J_d. The logarithm of λ is the energy derived from the change in agonist affinity that powers R↔R* gating.

Figure 2. Estimating E₂

A, a step to a high ACh concentration in an outside-out patch. The rapid activation (downward) reflects binding to R and A₂R↔A₂R* gating (boxed states, Fig. 1) and the slower decay phase reflects entry into desensitized states (mainly from A₂R*). Note the openings even after prolonged application. B, steady-state currents from a cell-attached patch (1 mm ACh). Channel openings (down) occur in clusters that represent the binding and gating activity of a single AChR. In the silent periods between clusters all AChRs in the patch are desensitized. C, the probability of being open within a cluster decreases with lower agonist concentrations because the un- and mono-liganded R states are increasingly occupied. Desensitization is removed from the analysis by fitting only intra-cluster interval durations by the ‘main’ part of the MWC scheme. E₂≍ 25 and K_d≍ 150 μm for ACh and wt adult mouse AChRs (23°C, −70 mV).

Figure 3. Estimating E₀

A, unliganded openings are rare in wt AChRs, but increase in frequency in AChRs with mutations that increase E₂. Combining mutations further increases unliganded openings, and with very high gain-of-function combinations leads to clusters that reflect the gating behaviour of a single channel in the absence of agonists. B, the predicted fold change in E₂ (assuming mutations only alter E₀ and have energetically independent effects) and the observed single-channel E₀ for the combinations are correlated. The extrapolated ‘observed’ value when the predicted fold change is unity is E₀^wt≍ 7 × 10⁻⁷.

Figure 4. Estimating E₁

A, expressing both wt and αW149M mutant subunits results in AChRs with either two, one or zero operational transmitter binding sites. B, currents cluster from AChRs with two (top traces) or one (bottom traces) functional binding site(s). The current amplitudes are smaller for choline because of channel block by this agonist. Both ACh and choline, at concentrations that lead to full occupation of the wt site and low occupation of the mutant site, generate mono-liganded current clusters. To assist the analysis, the AChRs had an independent gain-of-function background mutation, either αS269I (ACh) or αP272A (choline). For these agonists the two wt binding sites are equivalent and independent so only one population of mono-liganded clusters is apparent. After correcting for the background, E₂^ACh≍ 25 and E₂^choline≍ 0.046, and E₁^ACh≍ 0.0048 and E₁^choline≍ 1.6 × 10⁻⁴. Using the relationship E₀=E₁²/E₂ we estimate from both the ACh and choline results that E₀≍ 7 × 10⁻⁷.

Figure 5. The parameters of the MWC model for adult mouse neuromuscular AChRs

The rate constants are approximate (−70 mV, 23°C, ACh). The corresponding equilibrium constants are shown below. The coupling constant, λ= (E₂/E₀), is 6000 for ACh, which corresponds to an energy of −5.1 kcal mol ⁻¹ per ACh molecule.