Acetylcholine receptor gating at extracellular transmembrane domain interface: the cys-loop and M2-M3 linker

Abstract

Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a "gate" in the transmembrane domain (TMD). We used Phi-value analysis to probe the relative timing of the gating motions of alpha-subunit residues located near the ECD-TMD interface. Mutation of four of the seven amino acids in the M2-M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (K(eq)) by up to 10,000-fold (P272 > I274 > A270 > G275). The average Phi-value for the whole linker was approximately 0.64. One interpretation of this result is that the gating motions of the M2-M3 linker are approximately synchronous with those of much of M2 (approximately 0.64), but occur after those of the transmitter binding site region (approximately 0.93) and loops 2 and 7 (approximately 0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed K(eq) by 2800-, 10-, and 18-fold, respectively, and with an average Phi-value of 0.74, similar to those of other cys-loop residues. Even though V132 and I274 are close, the energetic coupling between I and V mutants of these positions was small (< or =0.51 kcal mol(-1)). The M2-M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an approximately 16-A border and involve about a dozen residues.

Figure 1.

The α_ɛ subunit M2–M3 linker (from 2bg9.pdb; Unwin, 2005). Left, in each AChR subunit the extracellular domain (ECD) is mostly β-sheet and the transmembrane domain (TMD) has four α helices. Residue W149 (brown) marks the transmitter binding site, M2 (blue) lines the pore and is linked to M3 (red) at the ECD/TMD interface (boxed). Right, expanded view of the boxed area. M2–M3 linker residues A270-K276 are colored by element: green, carbon; red, oxygen and blue, nitrogen. The colored domains are: orange, cys-loop; cyan, loop 2. M1, M4, loop9, and the preM1 linker have been removed for clarity. The core sequence of the M2–M3 linker is PLIG (272–275).

Figure 2.

Example single-channel current clusters for different M2-M3 linker constructs. (A) Continuous recording at low time resolution (I274F activated by 500 μM acetylcholine). Openings are down. The long gaps between clusters of opening reflect times when all channels in the patch are desensitized. Each cluster mainly reflects C↔O gating of a single AChR. Below, one cluster shown on an expanded time scale. (B) Example clusters of different constructs. Currents were activated either by 20 mM choline (indicated by a subscript) or by 500 μM ACh.

Figure 3.

Rate-equilibrium free energy relationships (REFERs) for all seven M2–M3 linker residues. Φ (indicated in the lower right corner of each plot; see Table II) is estimated as slope of a linear fit. The wild-type side chain is boxed. The agonist was either ACh (500 μM, closed circles) or choline (20 mM, open circles). Both the opening rate and equilibrium constants have been normalized (mutant/wt). Φ values could not be determined for positions V271, L273, and K276 because the change in K_eq was too small. The average Φ-value for the M2–M3 linker is ∼0.64.

Figure 4.

The mutation I274 shows wild-type binding properties. Interval durations were obtained at three different ACh concentrations (30, 50, and 100 μM) and the association and dissociation rate constants were estimated by fitting with a kinetic scheme that assumed two equal and independent transmitter binding steps followed by a single gating step (dead time = 35, 75, and 35 μs, respectively). Left, interval duration histograms and square-root probability density functions (solid lines) calculated from the globally optimized rate constants. Right, an example cluster at each concentration. Total number of events analyzed were 48,776. The optimal rate constants were: k₊ (single-site association) = 141 μM⁻¹s⁻¹, k_- (single-site dissociation) = 14,781 s⁻¹. We calculate K_d (k₋/k₊) = 105 μM. For comparison, one estimate for wild-type AChRs is k₊ = 169 μM⁻¹s⁻¹ and k₋ = 16,904 s⁻¹, K_d= 100 μM (Akk et al., 1996). There is no significant effect of this mutation on ACh binding to closed AChRs.

Figure 5.

Kinetic analysis of cys-loop residues. (A) Example clusters and REFER for αV132. The Φ-value was 0.75. (B) Example clusters and REFER for αT133. The change in K_eq was too small to allow an estimation of Φ. (C) Example clusters and REFER for αH134. The Φ-value was 0.71. (D) Example clusters and REFER for αF135. The Φ-value was 0.75. The wild-type side chain is boxed. The agonist was either ACh (500 μM, closed circles) or choline (20 mM, open circles).

Figure 6.

Cluster analysis of Φ values. The Φ values for the indicated α-subunit residues (in the M2 helix, M2–M3 linker, cys-loop, and loop 2) were grouped into populations by using a segmental k-mean algorithm (see Materials and methods). Inset, the sum squared deviation (SSQ) decreases sharply between 1 and 2 populations and gradually thereafter, indicating that the most likely number of Φ populations is two. The mean ± SEM values for these populations (dashed lines) are shown. Each residue was assigned either to the Φ = 0.77 population (open triangles) or the Φ = 0.63 population (filled circles). The error bars on Φ are ±SD. Y127 and residues in loop 2 and the cys-loop belong to the 0.77 population, and many residues in M2 and the M2–M3 linker belong to the 0.63 population. L2 and L7 are the mean Φ values for loop 2 and cys-loop residues measured by Chakrapani et al. (2004). Other sources: M2 (Mitra et al., 2005; Purohit et al., 2007), E45 (Purohit and Auerbach, 2007a), and Y127 (Purohit and Auerbach, 2007b).

Figure 7.

The ECD–TMD interface. The M2–M3 linker residues that were sensitive to mutation are labeled (P272 > I274 > G275 > A270). (A) Side view, from the middle of the ɛ-subunit. The lumen of the pore is to the right and the membrane bilayer is to the left.Orange, cys-loop (left) and loop 2 (right). Green, M2–M3 linker. Red, residue Y277. The N terminus of the M3 helix is Y277 and the C-terminus of the M2 helix is A270 (see Fig 1). (B) Radial view, from the bilayer.