The gating isomerization of neuromuscular acetylcholine receptors

Abstract

Acetylcholine receptor-channels are allosteric proteins that isomerize ('gate') between conformations that have a low vs. high affinity for the transmitter and conductance for ions. In order to comprehend the mechanism by which the affinity and conductance changes are linked it is of value to know the magnitude, timing and distribution of energy flowing through the system. Knowing both the di- and unliganded gating equilibrium constants (E(2) and E(0)) is a foundation for understanding the AChR gating mechanism and for engineering both the ligand and the protein to operate in predictable ways. In adult mouse neuromuscular receptors activated by acetylcholine, E(2) = 28 and E(0) approximately 6.5 x 10(7). At each (equivalent) transmitter binding site acetylcholine provides approximately 5.2 kcal mol(1) to motivate the isomerization. The partial agonist choline provides approximately 3.3 kcal mol(1). The relative time of a residue's gating energy change is revealed by the slope of its rate-equilibrium constant relationship. A map of this parameter suggests that energy propagates as a conformational cascade between the transmitter binding sites and the gate region. Although gating energy changes are widespread throughout the protein, some residues are particularly sensitive to perturbations. Several specific proposals for the structural events that comprise the gating conformational cascade are discussed.

Anthony Auerbach (State University of New York at Buffalo) received a PhD in neuroscience from the University of Oregon. He was a postdoctoral fellow with Jose del Castillo (synaptic physiology) and Fred Sachs (single-channel biophysics) before joining the faculty at SUNY Buffalo. The focus of his research has always been the neuromuscular acetylcholine receptor, with ancillary projects on NMDA receptors, GABA receptors and transition state theory. He is a co-developer of the QUB software suite for channel analysis and has received back-to-back Jacob Javits Investigator awards from NIH.

Figure 1. Energetics of the AChR gating isomerization

A, hypothetical energy landscape for the R↔R* isomerization. Inset, possible shapes of the separating energy barrier (k_B, Boltzmann constant; T, absolute temperature; k_BT= 0.6 kcal mol⁻¹ at 23°C). Top-to-bottom: a point, a parabola with continuum friction, a rugged landscape with metastable intermediates. B, the binding–gating cycle. Horizontal is binding and vertical is the isomerization. R and R*, stable structural ensembles having a low vs. high affinity for agonists and conductance; A, agonist; arrows represent barrier region events. Two binding steps have been collapsed into a single step. K_a, equilibrium association constant to R; J_a, equilibrium association constant to R*; E₀, unliganded isomerization equilibrium constant; E₂, diliganded equilibrium isomerization constant. Without any external energy, E₂/E₀= (J_a1J_a2)/(K_a1K_a2).

Figure 2. Unliganded AChR gating

A, spontaneous currents recorded without any agonists (R* is down). Mutations increase the unliganded isomerization equilibrium constant E₀. Constructs (top-to-bottom): wild-type, αY127F, αD97A, α(Y127F + D97A), α(Y127F + D97A + P272A). Bottom: unliganded AChRs enter states associated with desensitization. B, observed E₀ values for mutants are correlated with those expected from the fold-change in E₂. The slope of the dashed line is 1. C, R/E plot for ligands at the binding site: •, diliganded (different agonists); ○, unliganded. Equilibrium constants for ACh vs. no agonist represent a range-energy of 10.4 kcal mol⁻¹ (both α subunits combined). The slope of the line, Φ= 0.91.

Figure 3. Mono-liganded AChR gating

Cells were transfected with both wild-type and mutant α subunits and exposed to 500 μm ACh. The mutation (αW149M) prevents the binding site from using ligand-binding energy for the R↔R* isomerization (marked with an X). The expressed AChRs have either two (wt), one (hybrid) or zero functional binding sites (mut). Top: continuous, low resolution view; wt and hybrid openings are clustered. Bottom: higher resolution views. There is only one population of hybrid clusters because both isoforms have the same E₁.

Figure 4. Expanded binding–gating scheme

Top and bottom planes are binding steps, and connecting vertical lines are isomerization steps. Rate and equilibrium constants (boxed) pertain to the transmitter ACh (adult, mouse, neuromuscular AChRs, −100 mV, 23°C). The equilibrium dissociation constants (K_d and J_d) are shown for the α_ɛ binding site and the corresponding rate constants are shown for the αδ site. Dark arrow, a main pathway following a synaptic impulse.

Figure 5. Intermediate states

A, energy landscapes and corresponding simulated R/E plots showing that iso-energetic perturbations of the barrier region (grey lines) can yield different Φ values. The affinity change is the first event and the conductance change is the last. A mutation that changes the energy of an ‘early’ intermediate state alters most random walks across the barrier to give a high Φ value, whereas a ‘late’ perturbation gives a low Φ. The small, intermediate energy wells in the barrier region represent AChRs that have a high affinity and a low conductance. B, shut interval duration histograms from a broad barrier model simulation (CCCCO; all rates 5 μs⁻¹ except those from the end states which were 0.1 μs⁻¹). Top, at 1 GHz sampling many short-lived shut components (arising from sojourns in the barrier region intermediate states) are apparent (arrow, 5 μs). Bottom, same model at 1 MHz sampling (dead time, 2 μs). Only the exponential tail arising from the ensemble of intermediate states is apparent.

Figure 6. Maps of R↔R* energy changes

Left, side view of whole AChR (centre is ɛ subunit). Grey lines mark approximately the membrane (∼30 Å). In each subunit, the M2 helix lines the pore and M4 is at the periphery. Centre, α subunit histograms. Inset: there are most likely 4 Φ populations in the α subunit. Right, α subunit (viewed from ɛ subunit; * marks approximately the agonist location and arrow marks the gate region (M2–13′) position). Colours for range-energy and Φ as in histograms.

Figure 7. Events at the gate region of the pore

A, the pore-facing, equatorial residues 9′, 12′ and 13′ experience large and late energy changes, R→R* (blue ≥ 4 kcal mol⁻¹; red Φ < 0.4). Left, view from membrane; right, view from synapse. B, the energy changes at the equator may be caused both by the movement of the M2 helix (about midway through the isomerization) and by water entering this region at the end of the reaction. The R structure is from ELIC and the barrier and R* structures are from GLIC.