Distinct functional roles for the M4 α-helix from each homologous subunit in the heteropentameric ligand-gated ion channel nAChR

Abstract

The outermost lipid-exposed α-helix (M4) in each of the homologous α, β, δ, and γ/ε subunits of the muscle nicotinic acetylcholine receptor (nAChR) has previously been proposed to act as a lipid sensor. However, the mechanism by which this sensor would function is not clear. To explore how the M4 α-helix from each subunit in human adult muscle nAChR influences function, and thus explore its putative role in lipid sensing, we functionally characterized alanine mutations at every residue in αM4, βM4, δM4, and εM4, along with both alanine and deletion mutations in the post-M4 region of each subunit. Although no critical interactions involving residues on M4 or in post-M4 were identified, we found that numerous mutations at the M4-M1/M3 interface altered the agonist-induced response. In addition, homologous mutations in M4 in different subunits were found to have different effects on channel function. The functional effects of multiple mutations either along M4 in one subunit or at homologous positions of M4 in different subunits were also found to be additive. Finally, when characterized in both Xenopus oocytes and human embryonic kidney 293T cells, select αM4 mutations displayed cell-specific phenotypes, possibly because of the different membrane lipid environments. Collectively, our data suggest different functional roles for the M4 α-helix in each heteromeric nAChR subunit and predict that lipid sensing involving M4 occurs primarily through the cumulative interactions at the M4-M1/M3 interface, as opposed to the alteration of specific interactions that are critical to channel function.

Keywords: M4; channel gating; lipid sensing; lipid–protein interactions; nAChR; pentameric ligand-gated ion channels.

Figure 1

The M4 lipid sensors from each subunit of the nAChR are the most lipid-exposed TMD α-helices. Homology model of the human adult muscle nAChR based on the 2.7 Å resolution Torpedo nAChR structure (Protein Data Bank: 6UWZ). A, side view of the full model colored by domain with agonist-binding site residues (αTrp149) and channel gate residues (9′ and 13′) is shown as spheres colored cyan and tan, respectively. B, zoomed in view of a single subunit with the MA α-helix removed for clarity. The M4 α-helix, post-M4, and the Cys-loop are shown in red, blue, and green, respectively. C, top–down view of the TMD with M4 helices from each subunit colored red. nAChR, nicotinic acetylcholine receptor; TMD, transmembrane domain.

Figure 2

Functional effects of alanine mutations to residues within each M4 α-helix of the nAChR. Representative whole-cell two-electrode voltage clamp traces are shown for WT and the largest function-altering Ala mutants in the M4 α-helices of each subunit. Normalized concentration response curves for the selected mutants are shown in the bottom right. nAChR, nicotinic acetylcholine receptor.

Figure 3

Position of residues that cause significant changes in function when mutated to Ala. Zoomed in views of each subunit’s TMD with residues from M4 that significantly altered the EC₅₀ when mutated to Ala shown as sticks and colored according to residue type: aliphatic, tan; aromatic, yellow; polar/hydrogen bonding, green; negative, red; and positive, blue. A sequence alignment of M4 α-helices from each subunits of the human adult nAChR is shown at the bottom with residues colored according to residue type (aliphatic, black; aromatic, yellow; polar/hydrogen bonding, green; negative, red; and positive, blue) with post-M4 highlighted in gray. nAChR, nicotinic acetylcholine receptor; TMD, transmembrane domain.

Figure 4

Location of C-terminal deletions in each subunit. Side views of each subunit are shown with M4 helices and post-M4 semitransparent. Black spheres denote α-carbons for each deletion mutation.

Figure 5

Aromatic residues along the M4–M1/M3 interface in each subunit. Top–down (top) and side (bottom) views of each subunit’s TMD with aromatic residues at the M4–M1/M3 interface shown as yellow sticks. TMD, transmembrane domain.