Acetylcholine receptor gating: movement in the alpha-subunit extracellular domain

Abstract

Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains ("Phi blocks") move in staggering sequence to link an affinity change at the transmitter binding sites with a conductance change in the pore. In the alpha-subunit, the first Phi-block to move during channel opening is comprised of residues near the transmitter binding site and the second is comprised of residues near the base of the extracellular domain. We used the rate constants estimated from single-channel currents to infer the gating dynamics of Y127 and K145, in the inner and outer sheet of the beta-core of the alpha-subunit. Y127 is at the boundary between the first and second Phi blocks, at a subunit interface. alphaY127 mutations cause large changes in the gating equilibrium constant and with a characteristic Phi-value (Phi = 0.77) that places this residue in the second Phi-block. We also examined the effect on gating of mutations in neighboring residues deltaI43 (Phi = 0.86), epsilonN39 (complex kinetics), alphaI49 (no effect) and in residues that are homologous to alphaY127 on the epsilon, beta, and delta subunits (no effect). The extent to which alphaY127 gating motions are coupled to its neighbors was estimated by measuring the kinetic and equilibrium constants of constructs having mutations in alphaY127 (in both alpha subunits) plus residues alphaD97 or deltaI43. The magnitude of the coupling between alphaD97 and alphaY127 depended on the alphaY127 side chain and was small for both H (0.53 kcal/mol) and C (-0.37 kcal/mol) substitutions. The coupling across the single alpha-delta subunit boundary was larger (0.84 kcal/mol). The Phi-value for K145 (0.96) indicates that its gating motion is correlated temporally with the motions of residues in the first Phi-block and is not synchronous with those of alphaY127. This suggests that the inner and outer sheets of the alpha-subunit beta-core do not rotate as a rigid body.

Figure 1.

Location of αY127 in Torpedo AChRs. (A) A Cartoon of the α_ɛ/δ subunits viewed from the exterior of the AChR. Only the α_ɛ (left) and ɛ subunits are shown; the horizontal lines mark, approximately, the membrane. In α_δ the three Φ blocks that link the transmitter binding site with the gate are color coded as purple (Φ = 0.93, W149, K145), orange (Φ = 0.78, Y127), green (Φ = 0.65, S269), and red (Φ = 0.31, L251). (B and C) Expansion of boxed region in A. αK145 (purple) is on β-strand 7 and αY127 (orange) at the α_ɛ/ɛ (B) and α_δ/δ (C) subunit interface. αY127 is <4 Å from residues αD97 and αN94 in loop A (purple), αQ48 in loop 2 (orange), and ɛN39/δI43 in β-strand 1 (black). Structures were displayed by using PYMOL (DeLano Scientific).

Figure 2.

Example single-channel traces for 19 different side chains at αY127. (A) Continuous, low time resolution view of Y127F single-channel currents elicited by 20 mM choline (low pass filtered at 2 kHz for clarity; calibrations: 4 s, 2 pA). In the continued presence of such a high concentration of agonist, openings occur in clusters (open is down) separated by long nonconducting sojourns in “desensitized” states. Each cluster reflects C↔O gating of a single AChR. (B) Three gain-of-function constructs (F, W, and H) were activated by 20 mM choline and the current elicited for all other mutants were by 500 μM ACh. Example cluster for WT is shown for both the agonists. Calibration bars: (horizontal = 100 ms for choline and ACh, vertical scale bar = 2 pA, choline and 6 pA, ACh). (B) There was no apparent correlation of the side chain hydrophobicity or volume with the change in the diliganded gating equilibrium constant (K_eq). The r values were 0.26 (hydrophobicity) and 0.28 (volume).

Figure 3.

REFER analyses for αY127. Each point represents the mean of greater than two patches (Table I). Φ-Value was estimated as the slope of an unweighted linear fit to a log–log plot of normalized k_o vs. normalized K_eq for all 19 mutants. The slope Φ = 0.77 ± 0.02 makes αY127 a member of the second Φ-block that includes the cys-loop and loop 2. The open circles, filled circles, and open squares are choline, ACh, and carbamylcholine data points, respectively.

Figure 4.

The mutation αY127C does not alter the closed-channel equilibrium dissociation constant. Left, open and closed interval duration histograms at different ACh concentrations. The solid lines are calculated from the globally optimized rate constants. Number of events analyzed at various concentrations of ACh were: 100 μM, 3391; 300 μM, 2244; and 500 μM, 6940. Right, example clusters from each concentration. The optimal rate constants were: k₊ (single-site association) = 205 μM s⁻¹, k₋ (single-site dissociation) = 29604 s⁻¹, k_o = 2089 s⁻¹, and k_c = 5032 s⁻¹. We calculate K_d (=k₋/k₊) = 144 μM for the mutant. For comparison, the wt estimates are k₊ = 167 μM s⁻¹ and k_- = 24,745 s⁻¹, K_D = 148 μM (Chakrapani and Auerbach, 2005). There is no significant effect of this mutation on ACh binding to closed AChRs and we speculate that αY127 mutations that change K_eq do so by changing the unliganded gating equilibrium constant rather than the closed/open affinity ratio. Calibration bars for single channel traces: (horizontal scale bar = 100 ms, vertical scale bar = 6 pA).

Figure 5.

REFER analyses for δI43. Each point represents the mean of greater than two patches (Table IV). Φ-Value was estimated as the slope of an unweighted linear fit to a log–log plot of normalized k_o vs. normalized K_eq for all four mutants. The slope, Φ = 0.86 ± 0.10. Calibration bars for single channel traces: horizontal scale bar = 100 ms, vertical scale bar = 6 pA.

Figure 6.

REFER analyses for αK145. Each point represents the mean of greater than two patches (Table V). The Φ-value estimated for K145 is Φ = 0.96 ± 0.04. Calibration bars for single channel traces: horizontal scale bar = 100 ms, vertical scale bar = 6 pA.

Figure 7.

Gating movements occur mainly along the α/ɛ(δ) subunit interface. The structure is the mouse α-subunit ECD fragment (2qc1.pdb). The diliganded gating rate constants have been measured for the residues shown as spheres. White: mutations resulted in little or no change in K_eq (less than threefold), hence these residues show no indication of undergoing a gating motion (positions 20, 29, 31, 35, 40, 49, 54, 55, 56, 65, 110, 116, 120, 141, 143, 146, 207, and 208). Colored: mutations significantly changed K_eq. Purple, Φ ≈ 0.93 (positions 93, 95–100, 144, 145, 149, 152, 153, 190, 192, 198, and 200). Orange, Φ ≈ 0.77 (positions 45–48, 127, 132, 133, 134, 135, 137, 138, 140, and 209). The channel opening gating motions in the α-subunit ECD appear to propagate mainly along the interface with the ɛ (or δ) subunit, with residues near the binding site moving before those near the ECD–TMD interface.