Linking the acetylcholine receptor-channel agonist-binding sites with the gate

Abstract

The gating isomerization of neuromuscular acetylcholine receptors links the rearrangements of atoms at two transmitter-binding sites with those at a distant gate region in the pore. To explore the mechanism of this reversible process, we estimated the gating rate and equilibrium constants for receptors with point mutations of alpha-subunit residues located between the binding sites and the membrane domain (N95, A96, Y127, and I49). The maximum energy change caused by a side-chain substitution at alphaA96 was huge (approximately 8.6 kcal/mol, the largest value measured so far for any alpha-subunit amino acid). A Phi-value analysis suggests that alphaA96 experiences its change in energy (structure) approximately synchronously with residues alphaY127 and alphaI49, but after the agonist molecule and other residues in loop A. Double mutant-cycle experiments show that the energy changes at alphaA96 are strongly coupled with those of alphaY127 and alphaI49. We identify a column of mutation-sensitive residues in the alpha-subunit that may be a pathway for energy transfer through the extracellular domain in the gating isomerization.

Figure 1

Cyclic activation model for AChRs. Boxed letters are the stable end-states (R, low affinity and closed channel; R^∗, high affinity and open channel; A, the agonist). Arrows indicate the intermediate microstates (horizontal: ligand-binding; vertical: protein isomerization) that are too brief to be detected by our instrument. Equilibrium constants for each step: E, isomerization with zero, one, or two bound agonists; K_d, dissociation constant of R; J_d, dissociation constant of R^∗. For mouse, adult-type AChRs the two binding sites have approximately equal affinities for ACh (K_d = 140 mM and J_d = 20 nM). The energy between any two stable states is independent of the connecting pathway, hence E₂/E₀ = (K_d/J_d)².

Figure 2

Location and structure of residues. (A) Left: Torpedo AChR structure determined by cyroelectron microscopy (accession number 2BG9.pdb (13)). Shown as spheres in each of the two α-subunits are (blue) αW149 (at the transmitter-binding site) and (red) αI49 (loop 2), αA96 (loop A), and αY127 (β-strand 6). Horizontal lines approximately mark the membrane. The M2 transmembrane helix lines the ion permeation pathway and the M4 helix faces the lipids. Right: Mouse α-subunit extracellular domain fragment (accession number 2QC1.pdb (23)). A bound toxin molecule has been removed for clarity. The highlighted residues (red, bold) are αN95, αA96, αY127, and αI49. Five loops are color-coded: cyan, loop C; blue, loop B; yellow, loop A; tan, loop 2; green, loop 7 (cys loop). (B) Electron density map near the α(A96-Y127) region (from 2QC1.pdb). Left: Residues αA96 and αY127 are shown as sticks according to their position in 2QC1.pdb. Right: The same as the left except that αY127 is shown as an alternate rotamer, approximately filling an unoccupied region of the electron density map near αA96.

Figure 3

R/E constant analysis of αA96, αN95, αI49. (A) R/E plot for dilganded gating of AChRs with mutations at position αA96. The forward (channel-opening) isomerization rate and the gating equilibrium constants for each mutation were normalized by the wt value (triangle; Table 1). Solid circles: choline-activated; open circles: ACh-activated; ^∗, hybrid AChRs with only one mutant α-subunit. The slope of the line (Φ) is 0.79 ± 05 (SE). (B) Example currents clusters for each αA96 mutant construct (R^∗ is down).

Figure 4

Analysis of large gain-of-function αA96 mutants. (A) Continuous current trace from a cell transfected with αA96M plus wt α-, β-, δ-, and ɛ-subunit cDNAs. Three types of cluster are apparent, generated by wt (αA96+ αA96), double-mutant (αA96M+αA96M), and hybrid (αA96+αA96M) AChRs. (B) Histogram of inverse mean closed interval duration for all clusters in the patch shown in panel A. The three populations are (left to right) double mutant, hybrid, and wt. (C) R/E plot for clusters from the three populations shown in panel A. The gating equilibrium constant of the hybrid population is (on a log scale) about halfway between the wt and double-mutant populations, indicating that the αA96M mutation had approximately equal energetic effects in each subunit. (D) Spontaneous currents from the double αA96H mutant (no agonist in the bath or pipette). Each cluster reflects the gating activity of an individual AChR. The background construct had the mutation ɛL269T (in the M2 helix of the ɛ-subunit). (E) Example spontaneous clusters at higher resolution. (F) R/E plot for unliganded gating of αA96 mutants. The slope of the line (Φ) = 0.86 ± 0.20.

Figure 5

Single-channel currents and R/E plots for position αI49 and αN96. (A) αI49 mutant AChRs. Left: Example single-channel clusters for eight mutants, all activated by choline. Right: R/E plot. All substitutions except Ala increased the diliganded gating equilibrium constant. The slope of the line (Φ) = 0.71 ± 0.13. (B) αN95 mutant AChRs. Left: Example single-channel clusters for the W mutant (activated by ACh) and the Q mutant (activated by choline). Right: R/E plot. The slope of the line (Φ) = 0.86 ± 0.03.

Figure 6

Maps and histograms of range-energy and Φ in the α-subunit extracellular domain. (A) The range-energy is the natural logarithm of the largest/smallest gating equilibrium constant ratio for a family of mutations at each position. Blue spheres: ≥4.0 kcal/mol; ^∗ the approximate position of the agonist. The largest range-energy residues approximately form a column that links the transmitter-binding site (αW149) and the gate region of the pore-lining M2 helix (αV255). Bottom: Histogram of range-energy for all of the residues studied so far in the α-subunit. Residues in the marked bins: 1), W149, V261, S266, and I274; 2), V132, I264, and F135; 3), S268 and P265; 4), V255 and P272; and 5), E45. (B) Φ is the slope of the R/E relationship. Residues in the α-subunit with a range-energy >1.5 kcal/mol are shown as spheres. Color is according to the Φ-value in the histogram (for clarity, residues with Φ < 0.5 are not shown). αA96 and αP272 are surrounded by higher-Φ elements. αA96, αY127, and αE45 have similar Φ-values.