Tryptophan scanning of the acetylcholine receptor's betaM4 transmembrane domain: decoding allosteric linkage at the lipid-protein interface with ion-channel gating

Abstract

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that mediates fast excitatory synaptic transmission in the peripheral and central nervous systems. Changes in the structure and function of the AChR can lead to serious impairment of physiological processes. In this study, we combined site-directed mutagenesis, radioligand binding assays, electrophysiological recordings and Fourier analyses to characterize the functional role and structural aspects of the betaM4 transmembrane domain of the Torpedo AChR. We performed tryptophan replacements, from residues L438 through F455, along the betaM4 transmembrane domain. Expression levels of mutants F439W-G450W and F452W-I454W produced peak currents similar to or lower than those in wild-type (WT). Tryptophan substitutions at positions L438 and T451 led to a deficiency in either subunit expression or receptor assembly. Mutations L440W, V442W, C447W and S453W produced a gain-of-function response. Mutation F455W produced a loss of ion channel function. The periodicity profile of the normalized expression level (closed state) and EC(50) (open state) revealed a minor conformational change of 0.4 residues/turn of the betaM4 domain. These findings suggest that a minor movement of the betaM4 domain occurs during channel activation.

Figure 1

Sequence alignments of the βM4 domain and functional response of wild-type and βM4-mutant AChRs. (A) LFLYVFFVICSIGTGTFSIF (blue font) were examined in the current study. ***YV*FVICSI**FS** (highlighted in yellow and position number in green) are non-conserved residues among AChR species. Y*(441), F(443), F(444), C*(447) and S*(448) were labeled as lipid-exposed positions using photolabeling affinity [¹²⁵I]TID for the underline residues and [³H]DAF for the asterisks residues.^, The numbers at the bottom indicate the position in the Torpedo β1-subunit. (B) displays dose-response curves that were standardized to [¹²⁵I]-α-bungarotoxin (fmol) bound to AChR expressed on the oocyte surface membrane. The insert in each curve shows the dose-response curves normalized to maximum current. For the sake of clarity, we split the mutants in two groups (L438W to I446W and C447W to F455W). (C) shows representative families of macroscopic ionic current traces evoked by 1–300 µM ACh and recorded through two-electrode voltage clamp.

Figure 2

Periodicity profiles and Fourier transform of ACh EC₅₀ and AChR-normalized expression of the Torpedo tryptophan substitutions along the βM4 transmembrane domain. (A and C) show the periodicity profiles of the open-channel state (ACh EC₅₀ value) and closed-channel state (AChR-normalized expression), respectively. The values inside the boxes indicate the number of residues per helical turn between the adjacent maximums and minimums peaks. (B and D) represent the Fourier transform power spectrums from entire sequences of the AChR expression (fmol/ų) (closed-channel state) and ACh EC₅₀ (µM) (open-channel state) values that come from the tryptophan-scanning periodicity profiles data (A and C). Grey areas within the curves indicate the corresponding peak to average oscillation of the tryptophan-periodicity profiles in the closed- (D) and open-channel states (B).

Figure 3

Localization of the βM4-mutants AChR in the open and closed-channel state. (A–D) exhibit the helical net diagram of the amount of amino acid per helical turn between the adjacent maximum and minimum oscillatory peaks of the periodicity profile of the Torpedo AChR βM4 domain in the open-channel state (A and B) and the closed-channel state (C and D). The red dots represent the loss of function residues.

Figure 4

Spatial orientation of the AChR βM4 transmembrane domain. (A) display the βM4 domain along the helix. (B) shows the top view of the βM4 domain. (C) Illustrate the β M1–M4 domains. The amino acids in cyan color represent the gain in function positions. Asterisks (*) indicates the lipid-exposed positions using photolabeling affinity [¹²⁵I]TID (Y441, F443, I444, C447) and [³H]DAF (Y441, C447, S448). The 3D model representation is from the Unwin AChR 4Ź⁴ resolution structure interpretation using Pymol viewer (PDB code 2GB9).