From ab initio quantum mechanics to molecular neurobiology: a cation-pi binding site in the nicotinic receptor

Abstract

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation-pi interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation-pi binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues-tryptophan-149 of the alpha subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of alpha tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position alpha149 produces a constitutively active receptor.

Figure 1

Structural features of the nAChR. (Left) Global layout, adapted from data by Unwin (3), but with alternative ordering of subunits around the central axis (2, 20). (Right) Schematic of the agonist binding site (gray oval) viewed from the synapse, showing the large number of aromatic residues from three noncontiguous regions of the α subunit and key residues from the δ subunit also thought to contribute to binding. A comparable arrangement exists at the α/γ interface. Adapted from drawings previously presented by Galzi and Changeux (1) and Karlin and Akabas (2). Amino acid codes: W, tryptophan, Y, tyrosine, D, aspartate.

Figure 2

Structures of Trp derivatives incorporated by the nonsense-suppression method. Bta, benzothienylalanine; Np-Ala, naphthylalanine.

Figure 3

(A) Representative voltage-clamp current traces from oocytes expressing Trp (wild type, Left) and 5,7-F₂-Trp (Right) at α149. Bars represent application of ACh (μM). (B) Dose–response relations and fits to the Hill equation for (left to right): Trp; 5-F-Trp; 5,7-F₂-Trp; 5,6,7-F₃-Trp; and 4,5,6,7-F₄-Trp. EC₅₀ values are given in Table 2. (C) Plot of log[EC₅₀/EC₅₀(wild type)] vs. quantitative cation–π binding ability at α149 for the same residues as in B. Data are from Table 2. The data fit the line y = 3.2 − 0.096x, with a correlation coefficient r = 0.99. Error bars are approximately the size of the markers.

Figure 4

(Left) Schematic of the quaternary ammonium of ACh positioned over the 6-ring of αTrp-149. (Right) Structure of Tyr-O3Q, showing that the tethered quaternary ammonium can be positioned in roughly the same location as the quaternary ammonium of ACh.

Figure 5

(A) Standing inward currents and ACh responses in oocytes expressing nAChRs with Tyr-O3Q incorporated at α149 and a Leu-9′ → Ser mutation in M2 of the β subunit. The initial current of almost −3 μA, the standing current as a result of the mutant channels, is substantially eliminated by channel blockers such as QX-314 and TMB-8 or the cholinergic antagonist dTC. Application of ACh leads to increased currents. (B) ACh-induced desensitization affects the standing current. During the first application of TMB-8 (5 μM), the standing outward current was reduced reversibly to nearly zero. ACh (25 μM) was then added, inducing an inward current that desensitized. During the desensitization phase, the standing current was reduced. TMB-8 was added again during the desensitization phase. The TMB-8 deflection reached the former plateau level rather than producing a net outward current. These interactions show that ACh desensitizes the conductance mechanism that produces the standing current; therefore, the standing current is produced by functional nAChR. (C) Single-channel records and open-time distribution from receptors containing Tyr-O3Q at position α149. The traces show selected openings from cell-attached patches recorded at −80 mV. The open-time distribution from a typical patch is fitted (least-squares) to a single exponential decay with a time constant of 1.4 ms. Openings less than 0.6 ms were excluded from the analysis to eliminate contributions from the spontaneous openings of βLeu-9′ → Ser receptors, which have a time constant of 0.3 ms (H. Zhang et al., personal communication).