Sources of energy for gating by neurotransmitters in acetylcholine receptor channels

Abstract

Nicotinic acetylcholine receptors (AChRs) mediate signaling in the central and peripheral nervous systems. The AChR gating conformational change is powered by a low- to high-affinity change for neurotransmitters at two transmitter binding sites. We estimated (from single-channel currents) the components of energy for gating arising from binding site aromatic residues in the α-subunit. All mutations reduced the energy (TyrC1>>TrpB≈TyrC2>TyrA), with TyrC1 providing ~40% of the total. Considered one at a time, the fractional energy contributions from the aromatic rings were TrpB ~35%, TyrC1 ~28%, TyrC2 ~28%, and TyrA ~10%. Together, TrpB, TyrC1, and TyrC2 comprise an "aromatic triad" that provides much of the total energy from the transmitter for gating. Analysis of mutant pairs suggests that the energy contributions from some residues are nearly independent. Mutations of TyrC1 cause particularly large energy reductions because they remove two favorable and approximately equal interactions between the aromatic ring and the quaternary amine of the agonist and between the hydroxyl and αLysβ7.

Fig. 1.

Transmitter binding site. (A) Torpedo AChR (PDB 2bg9) (2). The asterisk indicates a transmitter binding site. (B) Aromatic residues in the Lymnaea AChBP (PDB 1uv6) (32). Loops: A, pink; B, green; and C, tan. Carbamylcholine (CCh), white. (C) Contributions from the side chains. The numbers are energies (kcal/mol) from the corresponding chemical moiety. TrpB, TyrC1, and TyrC2 comprise an “aromatic triad” that contributes most of the total energy. One exit path for energy is TyrC1-Lysβ7.

Fig. 2.

Mutations of TrpB. (A) Diliganded gating. (Upper) Low-time-resolution view of TrpB-F currents (140 mM ACh, +100 mV). Openings (up) occur in clusters, each from one AChR. (Lower) Intracluster opening frequency increases with increasing [ACh] to reach at plateau (arrow, 30 mM ACh). (Inset) Example clusters (TrpB-F). (B) Unliganded gating. (Upper) Low-time-resolution view of unliganded TrpB-F currents (−100 mV). Openings (down) occur in clusters even in the absence of any agonists. (Lower) E₀ for each mutation on a log scale. The H and M mutations decreased, and all others increased, E₀^wt. Traces are examples of unliganded current clusters.

Fig. 3.

Energies of the aromatic mutants. In wt AChRs, ΔG_B^ACh = −5.1 kcal/mol (topmost bar). All mutations of all the residues reduced ΔG_B^ACh. Mutations of TyrC1 caused the largest reductions, and those of TyrA caused the smallest reductions. The error limits on the ΔG_B^ACh values are given in Table S1.

Fig. 4.

Φ-Analysis. The rate and equilibrium constants were normalized by the wt values. Each symbol is a different mutant (average of ≥3 patches). Filled circles (●) illustrate diliganded ACh gating, and open triangles (△) illustrate gating without any agonists. The slopes (Φ) are somewhat higher with ACh compared with without any agonists.

Fig. 5.

Relative effects of mutations. (A) Change in ΔG_B^ACh for substitutions common to all four aromatic residues. For each substitution, the loss of energy (positive; rightward) was largest for TyrC1 (black) in all cases. The Ala substitution caused the largest energy loss at all positions except TyrA. (B) Energy changes for different substitutions. (Top to Bottom) Removing the aromatic group had the largest effect at TrpB, swapping the aromatic group (F ↔ W) had little effect at TyrC2 and TyrA, and removing the OH group had the largest effect at TyrC1.