Acetylcholine receptor gating at extracellular transmembrane domain interface: the "pre-M1" linker

Abstract

Charged residues in the beta10-M1 linker region ("pre-M1") are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational "wave." We examined the effects of mutations to all five residues in pre-M1 (positions M207-P211) plus E45 in loop 2 in the mouse alpha(1)-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (K(eq)), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in K(eq) (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Phi values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Phi values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only -0.33 kcal/mol (for both alpha subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.

Figure 1.

The AChR pre-M1 linker. (A) The Torpedo AChR (2bg9.pdb). The pre-M1 linker in the α_ɛ subunit is highlighted (black spheres). Horizontal lines and arrow mark, approximately, the membrane and the transmitter binding site. ECD, extracellular domain, TMD, transmembrane domain. (B) The five pre-M1 residues, between β-strand 10 in the ECD (tan) and M1 in the TMD (gray), plus residue E45 in loop 2 are colored by element (carbon, green; nitrogen, blue; oxygen red). For clarity, loop 9 and M4 are not shown. L7 is loop 7 (the “cys-loop”) and L2 is loop 2. The pre-M1 sequence (positions 207–211) for Torpedo is MQRIP. Position 210 is L in mouse. Structures were displayed by using PYMOL (DeLano Scientific).

Figure 2.

E45 mutational series. (A) Low time resolution view showing clusters of single-channel openings (E45R activated by choline; open is down). (B) Higher time resolution view of clusters for different side chains. Left, activated by 20 mM choline and right, activated by 500 μM ACh. The relationship between the change in the diliganded gating equilibrium constant (K_eq) and the chemical nature of the side chain is not simple. (C) REFER analysis. Each point represents the mean of multiple patches (Table I) (wt is boxed; filled/open circles are ACh/choline-activated). The Φ-value (bottom right) was estimated as the slope of a linear fit to a log–log plot of normalized k_o vs. normalized K_eq for all 10 mutants. E45 has the same Φ-value as other residues in loop 2, along with αY127 and residues in loop 7.

Figure 3.

R209 mutational series. (A) R209 mutants on a wt background. Top, example single-channel clusters activated by choline or ACh. Single-channel currents were not detected for R209A, C, D, E, F, G, I, L, M, N, P, S, T, V, W, Y, and the deletion mutant. Bottom, each point in the REFER plot represents the mean of multiple patches (Table III); wt is boxed. The error limit on Φ (bottom right) was large (±0.16). (B) R209 mutants on the δL265T background that specifically slows channel closing. Top, example single-channel clusters activated by choline. Bottom, REFER plot. The error limit on Φ was large (±0.19)

Figure 4.

L210 mutational series. (A) Example clusters elicited by choline. All of the mutations increased K_eq and the cluster open probability, mainly by prolonging the open time (slowing the closing rate constant). (B) REFER plot (Table IV); wt is boxed. The Φ-value for L210 was Φ = 0.35 ± 0.12, which suggests that this residue moves “late” in the gating isomerization.

Figure 5.

Energy coupling between R209 and E45. Example currents and REFERs for R209 mutations series on three different E45 backgrounds: (A) E45R, (B) E45L, and (C) E45A (Table III). Mutations were made at both R209 and E45 (in both α subunits) and current was activated by choline (open circles) or ACh (filled circles). The R209A and R209N point mutants in a wt background did not yield functional AChRs but did so in the E45R and E45L backgrounds. The Φ values estimated for position 209 in E45R and E45L backgrounds are imprecise because of a large SD (±0.23 and ±0.26). A mutant cycle analysis for these constructs indicates that the coupling between R209K/45A, R209Q/E45R, and R209H/E45R is greater than between R209Q/E45A and R209K/E45L (Table III). The REFER for R209 on an E45A background was linear over a greater than three order of magnitude range in K_eq, and is our best estimate of the Φ-value for R209.