Gating dynamics of the acetylcholine receptor extracellular domain

Abstract

We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in the extracellular domain (ECD) of the alpha-subunit of mouse muscle-type acetylcholine receptors (AChRs). The crystal structure of an acetylcholine binding protein (AChBP) suggests that the ECD is comprised of a beta-sandwich core that is surrounded by loops. Here we focus on loops 2 and 7, which lie at the interface of the AChR extracellular and transmembrane domains. Side chain substitutions in these loops primarily affect channel gating by either decreasing or increasing the gating equilibrium constant. Many of the mutations to the beta-core prevent the expression of functional AChRs, but of the mutants that did express almost all had wild-type behavior. Rate-equilibrium free energy relationship analyses reveal the presence of two contiguous, distinct synchronously-gating domains in the alpha-subunit ECD that move sequentially during the AChR gating reaction. The transmitter-binding site/loop 5 domain moves first (Phi = 0.93) and is followed by the loop 2/loop 7 domain (Phi = 0.80). These movements precede that of the extracellular linker (Phi = 0.69). We hypothesize that AChR gating occurs as the stepwise movements of such domains that link the low-to-high affinity conformational change in the TBS with the low-to-high conductance conformational change in the pore.

Figure 1.

Location of loops 2, 5, 7, and the β-sandwich core in AChBP. The AChBP subunit interface is shown perpendicular to the pseudo-fivefold axis of symmetry. A HEPES molecule in the transmitter binding site is shown in orange. The AChR α-subunit corresponds to the “+”-side of this interface (cyan). Loop 2 and 7 are at the interface of the extracellular and transmembrane domains.

SCHEME I

Figure 2.

αV46 in loop 2: single-channel currents. For each panel the top three current traces are continuous and the boxed region is shown below at a higher time-resolution (30 μM ACh; open is down; filtered at 10 kHz for display). All mutations decreased the cluster P_open except I. The effects on cluster P_open were mainly due to a change in the lifetime of closed-intervals (Table II).

Figure 3.

αV46: kinetic analysis across concentrations. (A) αV46M. (B) αV46Y. For each construct single-channel currents (left) and interval duration histograms (right) elicited by different ACh concentrations (30, 50, and 70 μM, top to bottom) are shown. The solid lines are probability density functions calculated from the rate constants of Scheme I obtained from a joint fit across all concentrations. Neither of these mutations had a significant effect on agonist binding to closed AChRs (Table III).

Figure 4.

αN47 in loop 2: single-channel currents. For each panel the top three current traces are continuous and the boxed region is shown below at a higher time-resolution. The L and D mutations (30 μM ACh) decreased the cluster P_open, whereas the A and K mutations (20 mM choline) increased the cluster P_open (filtered at 2 kHz for display). For both classes of mutation the effect on cluster P_open was mainly due to a change in the closed-interval lifetime (Table II).

Figure 5.

αN47D: kinetic analysis across concentrations. Representative clusters and dwell time histograms, with superimposed probability density functions calculated from the rate constants of Scheme I obtained from a joint fit across all concentrations. This mutation had no effect on ACh association or dissociation to closed AChRs (Table III).

Figure 6.

The diliganded gating equilibrium constant is not strongly correlated with the chemical properties of the sidechains at αN47 and αV46. (A) At positions αN47 there is no correlation between side-chain hydrophobicity or volume and the effect on the diliganded gating equilibrium constant (L_2.). (B) At positions αV46, there is some apparent correlation between side-chain hydrophobicity and L₂, which decreases with increasing side-chain polarity.

Figure 7.

αQ48 in loop 2: single-channel currents. (A) Example clusters elicited by 30 μM ACh. The αQ48K substitution decreased the cluster P_open mainly by increasing the closed-interval lifetimes. The αQ48A substitution increased the cluster P_open, but both the closed and open interval lifetimes decreased (a “catalytic” effect). (B) Representative clusters from wt and αQ48A AChRs at low and high concentrations of choline. The A substitution increases L₂ (Table II).

Figure 8.

αQ48K: kinetic analysis across concentrations. Representative clusters and dwell time histograms, with superimposed probability density functions calculated from the rate constants of Scheme I obtained from a joint fit across all concentrations. This mutation did not have a significant effect on ACh association or dissociation to closed AChRs (Table III).

Figure 9.

Loop 7: single-channel currents. For each panel the top three current traces are continuous and the boxed region is shown below at a higher time-resolution. These mutations decreased cluster P_open (30 μM ACh) mainly by increasing the closed-interval lifetimes (Table V).

Figure 10.

αH134S: kinetic analysis across concentrations. Representative clusters and dwell time histograms, with superimposed probability density functions calculated from the rate constants of Scheme I obtained from a joint fit across all concentrations. The αH134S mutant had no significant effect on agonist binding (Table VI).

Figure 11.

αF135A and αQ140A in loop 7 exhibit two open states. Example clusters elicited by the indicated ACh concentrations and their corresponding interval duration histograms. The histograms for the open-interval dwell times show two distinct components. The closed- and -open interval dwell times for all the concentrations were fitted jointly by a modified version of Scheme I in which a second open state was connected to the diliganded-closed state. Neither of these mutations had any significant effect on agonist binding (Table VI).

Figure 12.

αM144: single-channel currents. Continuous traces showing clusters elicited by 30 μM ACh, with the boxed cluster shown below at a higher time-resolution. This residue is on strand β7 and is close to the disulfide bond that binds the β-sandwich core. αM144L increased cluster P_open while αM144S decreased cluster P_open (Table VII).

Figure 13.

Rate-equilibrium free energy relationships. The y-axis is the log of the channel-opening rate constant, and the x-axis is the log of the diliganded-gating equilibrium constant. The Φ-values are the slopes of the linear fits ± SD. In all panels, the wt value is shown as an open circle. (A) The overall Φ-value for loop 2 is 0.81, which indicates that during diliganded opening this domain moves after the TBS and loop 5 (Φ = 0.93) but before residue αS269 (Φ = 0.69) in the EL/M2. (B) The Φ-value for the main gating reaction for loop 7 is 0.78. This value is indistinguishable from that of loop 2, thus these two domains move synchronously in the diliganded gating reaction. (C) The Φ-value for position αM144 is 0.84. We suspect that this residue moves in synchrony with loop 2 and 7. (B) The Φ-value for the secondary gating reaction for loop 7 is 0.37, which indicates that this domain moves relatively late in this reaction.

Figure 14.

A Φ-map of the AChR. A model of the AChR (α- and δ-subunits) with residues color-coded according to their Φ-values for diliganded gating. The AChR is organized into contiguous, discrete, and synchronous domains that move sequentially, each as a rigid body, during gating. During channel opening, the TBS/loop 5 gating domain is the first to undergo a conformational change (red; Φ = 0.93), followed by the loop 2/loop 7/M144 gating domain (yellow; Φ = 0.80), followed by residue αS269 in the extracellular linker/αM2 (in green; Φ = 0.69), followed by the upper part of δM2 (blue; Φ = 0.32), and then the lower half of δM2 (magenta; Φ = 0.0). The extracellular domain is AChBP (Brejc et al., 2001) and the membrane domain is from Torpedo AChR (Miyazawa et al., 2003). The residues whose Φ-values were estimated independently are (Akk et al., 1996, 1999; Grosman et al., 2000a,b; Cymes et al., 2002; Chakrapani et al., 2003): αTBS (D200, D152, Y93, W149, Y198), αloop 5 (L92, Y93, N94, N95, A96, D97, G98, D99, F100), αloop 2 (V46, N47, Q48), αloop 7 (H134, F135, F137, D138, Q140, N141), rest of the αECD (L40, A122, M144), and δM2 (S258, I261, V263, L265, A266, S268, V269, L272, S274). Residues between those with similar Φ-values were given the same color code even though they were not measured experimentally.