The functional role of the αM4 transmembrane helix in the muscle nicotinic acetylcholine receptor probed through mutagenesis and coevolutionary analyses

Abstract

The activity of the muscle-type Torpedo nicotinic acetylcholine receptor (nAChR) is highly sensitive to lipids, but the underlying mechanisms remain poorly understood. The nAChR transmembrane α-helix, M4, is positioned at the perimeter of each subunit in direct contact with lipids and likely plays a central role in lipid sensing. To gain insight into the mechanisms underlying nAChR lipid sensing, we used homology modeling, coevolutionary analyses, site-directed mutagenesis, and electrophysiology to examine the role of the α-subunit M4 (αM4) in the function of the adult muscle nAChR. Ala substitutions for most αM4 residues, including those in clusters of polar residues at both the N and C termini, and deletion of up to 11 C-terminal residues had little impact on the agonist-induced response. Even Ala substitutions for coevolved pairs of residues at the interface between αM4 and the adjacent helices, αM1 and αM3, had little effect, although some impaired nAChR expression. On the other hand, Ala substitutions for Thr422 and Arg429 caused relatively large losses of function, suggesting functional roles for these specific residues. Ala substitutions for aromatic residues at the αM4-αM1/αM3 interface generally led to gains of function, as previously reported for the prokaryotic homolog, the Erwinia chrysanthemi ligand-gated ion channel (ELIC). The functional effects of individual Ala substitutions in αM4 were found to be additive, although not in a completely independent manner. Our results provide insight into the structural features of αM4 that are important. They also suggest how lipid-dependent changes in αM4 structure ultimately modify nAChR function.

Keywords: Cys-loop receptor; ELIC; GLIC; M4; M4 transmembrane helix; channel gating; coevolutionary analysis; lipid sensing; lipid–protein interactions; membrane protein; mutagenesis; nicotinic acetylcholine receptor (nAChR); pentameric ligand-gated ion channel (pLGIC); protein evolution; transmembrane domain.

Figure 1.

Structure of the human αM4. A side view of the 2.7-Å cryo-EM structure of the Torpedo nAChR (PDB entry 6UWZ) is shown on the left. A zoomed-in view of the TMD of a human αM4 homology model, based on the 6UWZ structure, is shown on the right in two different orientations. Side chains are shown in ball and stick representation colored according to residue type: aromatic, yellow; polar/hydrogen bonding, green; positive, blue; negative, red; aliphatic, tan). Sequences of the M1, M3, and M4 helices from the human α1 nAChR are shown along the top with the helical regions boxed.

Figure 2.

Coevolved residues on αM4 and αM1 or αM3. Coevolved residues on αM4-αM1 (A and B) and αM4-αM3 (C and D) are mapped onto the 2BG9 Torpedo (A and C) and the 6UWZ Torpedo (B and D) homology models of the human muscle α subunit. Each panel shows a top view of the TMD from the extracellular surface (top) and a side view from within the membrane (bottom). Residues are shown as ball and stick representation and colored according to the coevolved residue pairs: Ile219-Val425, brown; Gly230-Leu411, cyan; Phe233-Phe414, purple; Tyr234-Leu411, cyan; Tyr277-Thr422, dark blue; Phe280-Gly421, red; Thr281-Cys418, green; Phe284-Val417, yellow; and Thr298-Met406, black.

Figure 3.

Functional effects of Ala substitutions for residues on αM4. Representative two-electrode voltage clamp whole-cell responses to different concentration traces are shown for select mutations. Dose-response relationships for select mutations are plotted on the bottom right.

Figure 4.

Comparison of the αM4 Ala scan heat map for the nAChR to those of GLIC and ELIC. The changes in EC₅₀ values resulting from Ala mutation of each residue on M4 is heat mapped onto the structure for GLIC (PDB entry 4HFI) (A), nAChR (6UWZ Torpedo model) (B), and ELIC (homology model based on the GLIC structure) (C). Residues on M4 are represented as spheres colored according to the magnitude of the change in EC₅₀. The scale runs between a 10-fold gain of function (red) and a 10-fold loss of function (blue), with no change in function shown as white. A mutant that gave no functional expression is shown in black, whereas a mutant that drastically altered desensitization kinetics is shown in dark gray.

Figure 5.

Aromatic residues at the αM4–αM1/αM3 interface. Aromatic residues at the αM4–αM1/αM3 interface are shown in ball and stick representations and are colored yellow. The TMDs from the 2BG9 Torpedo (left) and 6UWZ Torpedo (right) models are shown in both top views from the extracellular surface (top) and side views from within the membrane (bottom).