Design and control of acetylcholine receptor conformational change

Abstract

Allosteric proteins use energy derived from ligand binding to promote a global change in conformation. The "gating" equilibrium constant of acetylcholine receptor-channels (AChRs) is influenced by ligands, mutations, and membrane voltage. We engineered AChRs to have specific values of this constant by combining these perturbations, and then calculated the corresponding values for a reference condition. AChRs were designed to have specific rate and equilibrium constants simply by adding multiple, energetically independent mutations with known effects on gating. Mutations and depolarization (to remove channel block) changed the diliganded gating equilibrium constant only by changing the unliganded gating equilibrium constant (E(0)) and did not alter the energy from ligand binding. All of the tested perturbations were approximately energetically independent. We conclude that naturally occurring mutations mainly adjust E(0) and cause human disease because they generate AChRs that have physiologically inappropriate values of this constant. The results suggest that the energy associated with a structural change of a side chain in the gating isomerization is dissipated locally and is mainly independent of rigid body or normal mode motions of the protein. Gating rate and equilibrium constants are estimated for seven different AChR agonists using a stepwise engineering approach.

Fig. 1.

(Left) Agonists (full name given in SI Text). (Right) Torpedo AChR (accession number 2bg9.pdb). Transmitter binding site residues αW149 are blue and mutated residues are red. The mutated amino acids are scattered throughout the protein but are not at the transmitter binding sites. Another view and separate background constructs are shown in Fig. S2.

Fig. 2.

Gating of AChRs activated by DMP (reference conditions: WT α₂βδϵ, approximately -100 mV, 23 °C). (A) Clusters of openings that each reflects binding and gating activity of an individual AChR (open is down). (B) Higher resolution view of clusters at different [DMP]. (C) Intracluster interval duration histograms at different [DMP]. The solid lines are the global fit across concentrations (Scheme S1; K_d = 1.9 mM) with the gating rate constants of the B1 background (Fig. 3C). (D) Effective opening rate (f*) as a function of [DMP]. Solid line is the Hill equation using the opening rate constant of the B1 background (Fig. 3D). (E) At this membrane voltage the single-channel current amplitude (I_[A]) decreases with increasing [DMP] because of fast channel block by agonist molecules.

Fig. 3.

Gating of ϵS450W AChRs activated by DMP (+70 mV; construct B1). (A) Clusters of openings reflect binding and gating activity of individual AChRs (open is up). (B) Higher resolution view of clusters at different [DMP]. (C) Intracluster interval duration histograms at different [DMP]. The solid lines are the global fit to Scheme 1 (K_d = 2.1 mM, E₂ = 0.38). Arrows mark the time constants of the intervals at 5 mM DMP, reference condition (Fig. 2C). (D) The effective opening rate reaches a plateau at approximately 30 mM (f₂ = 818 s^-1). (E) Depolarization removes channel block so the single-channel current amplitude (I_[A]) is constant ≤ 100 mM DMP.

Fig. 4.

The effect of background mutations is similar for different agonists. In each plot the fold-change in the gating equilibrium constant E₂ caused by the mutation is given relative to the the B1 background (Table 1 and Table S2).

Fig. 5.

Gating of AChRs activated by ACh (+70 mV; B4 background). (A) Clusters of openings reflect binding and gating activity of individual AChRs (open is up). (B) Higher resolution view of clusters at different [ACh]. (C) Intracluster interval durations; solid lines are the global fit to Scheme S1 (K_d = 166 μM). (D) The effective opening rate reaches a plateau at approximately 1 mM. (E) The single-channel current amplitude (I_[A]) is constant because there is no channel block.

Fig. 6.

Rate–equilibrium (R–E) analysis of agonists. The opening rate constant (f₂) is plotted vs. the gating equilibrium constant (E₂) on a log-log scale. The parameters (Table 1) pertain to the B1 and reference conditions (WT AChRs, approximately -100 mV, 23 °C). The slope of the linear fit (Φ) is approximately 1.