Dynamics of the acetylcholine receptor pore at the gating transition state

Abstract

Neuromuscular acetylcholine receptors (AChRs) are ion channels that alternatively adopt stable conformations that either allow (open) or prohibit (closed) ionic conduction. We probed the dynamics of pore (M2) residues at the diliganded gating transition state by using single-channel kinetic and rate-equilibrium free energy relationship (phi-value) analyses of mutant AChRs. The mutations were at the equatorial (9') position of the alpha, beta, and epsilon subunits (n = 15) or at sites between the equator and the extracellular domain in the alpha-subunit (n = 8). We also studied AChRs having only one of the two alpha-subunits mutated. The results indicate that the alpha-subunit, like the delta-subunit, has a region of flexure near the middle of M2, that the two alpha-subunits experience distinct energy barriers to gating at the equator (but not elsewhere), and that the collective subunit motions at the equator are asymmetric during the AChR gating isomerization.

Fig. 1.

Kinetic analyses of α9′ double mutants. (A) Locations of residues in the α_ε-subunit [Protein Data Bank ID code 2bg9 (28)]. Top to bottom: W149 is at the transmitter binding site, and S269(27′), V259(17′), and L251(9′) are in the M2 segment. The arrow (30 Å) marks approximately the membrane and the pore. (B) Continuous, low-time resolution view of currents from AChRs having a L9′T mutation in both α-subunits (inward current, down). Boxed cluster is shown at higher time resolution in C.(C) Clusters of openings from AChRs having different 9′ mutations in both α subunits. The WT residue (Leu) is boxed. (D) REFER plots for the α9′ double-mutant series. The solid line/filled symbols pertain to a membrane potential of –100 mV, and the dashed line/open symbols pertain to a membrane potential of +60 mV (see Fig. 7).

Fig. 2.

Kinetic analyses of α17′ double-mutants. (A) Example clusters of α17′ double mutants activated by a saturating concentration of choline (20 mM). Open is downward. The WT residue (Val) is boxed. All mutations increased the cluster open probability by increasing the opening rate constant and decreasing the closing rate constant (Table 1). The kinetics for the small side chains (G, A, and S) appear by eye to be faster than the others, which we attribute to a catalytic effect (15, 18). (B) REFER plot for the α17′ mutant series. The Φ-value for the whole series is of 0.63 ± 0.18 (solid line). Eliminating the G, A, and S data points does not change significantly the Φ-value (0.61 ± 0.04; dashed line).

Fig. 3.

Kinetic analyses of α27′ double-mutants and hybrid. (A) Clusters of openings from AChRs having a Ser → Ile mutation at the α-subunit M2 27′ position (α269). Within a single patch there are three kinetically distinct cluster populations. (B) The cluster open probability distribution has three components that reflect the activity of AChRs having two (double-mutant), one (hybrid), and zero (WT) mutations. (C) REFER plot for α27′. The two α27′ positions make equal energetic contributions to and have progressed to the same extent at the transition state of the gating reaction (Φ = 0.65 ± 0.06).

Fig. 4.

Kinetic analyses of α9′ hybrids. (A) Clusters of openings from AChRs having two, one, or zero α9′ mutation (s). For each mutation, within a single patch there are four cluster populations having distinct kinetics and current amplitudes. The intermediate two populations reflect AChRs that are hybrids, having a point mutation in either α_ε or α_δ.(B) Current amplitude histogram for one patch (αL9′T). The hybrids each have distinct amplitudes because of differential equilibrium channel-block by the agonist (Table 1). (C) REFER plots for the hybrid populations. The WT side chain (Leu) is boxed. At the αM2–9′ position each hybrid has a distinct Φ-value, indicating that the two α-subunits have progressed to different extents at the transition state of the gating reaction.

Fig. 5.

REFERs for ε- and β-subunit equatorial (9′) residues. The WT side chain (Leu) is boxed. These positions have the same Φ-value, which suggests that they have progressed to the same extent at the transition state of the diliganded gating reaction. One α-subunit and the upper half of δM2 have similar Φ-values (≈0.32 and 0.34, respectively), whereas the other α-subunit has a Φ-value of 0.02.

Fig. 6.

Structural correlates of the Φ-values. (A) Longitudinal projection of the AChR showing four gating domains in the α_δ- and δ-subunits. The domains are color-coded by Φ-value: orange, ≈0.8; yellow, ≈0.65; red, ≈0.35; blue, ≈0.0. There is a longitudinal gradient in Φ-value, with residues organized into domains that appear to have distinct boundaries. For all domains, Φ-values of the residues at the boundaries (e.g., 2′ and 10′ in δM2) have been determined experimentally. (B) Symmetry of α-subunit Φ-values in hybrid constructs. Top to bottom: αD97 [loop 5; Φ = 0.93 (34)], αS269 (M2–27′, Φ = 0.65), αC418 [M4–10′, Φ = 0.50 (16)], and αL251 (M2–9′; Φ = 0.32 and 0.02). Early in the gating reaction the α-subunit gating motions are synchronous (green) but are asynchronous at the equator of M2 (red and blue). (C) Cross section of the pore at the equator showing the 9′ residues of all subunits (Leu) and the 12′ residue of the δ-subunit (Ala). The red atoms (Φ≈ 0.35) move in advance of the blue atoms (Φ≈ 0) in the diliganded AChR gating reaction. [Protein Data Bank ID code 2bg9 (28); pymol (http://pymol.sourceforge.net)].