Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors

Abstract

Nicotinic acetylcholine receptors (nAChR) are members of the Cys-loop ligand-gated ion channel superfamily. Muscle nAChR are heteropentamers that assemble from two α, and one each of β, γ, and δ subunits. Each subunit is composed of three domains, extracellular, transmembrane and intracellular. The transmembrane domain consists of four α-helical segments (M1-M4). Pioneering structural information was obtained using electronmicroscopy of Torpedo nAChR. The recently solved X-ray structure of the first eukaryotic Cys-loop receptor, a truncated (intracellular domain missing) glutamate-gated chloride channel α (GluClα) showed the same overall architecture. However, a significant difference with regard to the vertical alignment between the channel-lining segment M2 and segment M3 was observed. Here, we used functional studies utilizing disulfide trapping experiments in muscle nAChR to determine the spatial orientation between M2 and M3. Our results are in agreement with the vertical alignment as obtained when using the GluClα structure as a template to homology model muscle nAChR, however, they cannot be reconciled with the current Torpedo nAChR model. The vertical M2-M3 alignments as observed in X-ray structures of prokaryotic Gloeobacter violaceus ligand-gated ion channel and GluClα are in agreement. Our results further confirm that this alignment in Cys-loop receptors is conserved between prokaryotes and eukaryotes.

Fig 1

Multiple sequence alignments of Torpedo, muscle nAChR and GluCl α-subunits as obtained with ClustalW, and schematic nAChR representation. (a) Only the alignment comprising the transmembrane segments M2 and M3 is shown. Note that α-subunits of mouse nAChR and Torpedo are identical for 61 out of the shown 65 residues. The α-helical content as in the Torpedo structural model (PDB# 2BG9) is depicted with a grey bar on top of, and as in the GluCl structure (PDB# 3RHW) with a white bar below the sequences. For the M2 segment the position we mutated to Cys, 12’ corresponding to αT254 is indicated by bold font and underlined, and for orientation also 0’ and 20’ are marked by a light and dark grey box, respectively, and additionally the respective numbering. Positions mutated to Cys in M3 are indicated by bold font and by a continuous line for the GluCl-based set (277-YMLFTMV) and by a dotted line for the Torpedo-based set (288-SIIITV). (b) Subunit arrangement of muscle nAChR with a schematic representation of transmembrane segments (M1–M4) for the two α-subunits, and the potential cross-link schematically indicated in purple for the studied α-subunits. Subunits are labeled with greek letters, α, β, γ, andδ. (c) Muscle nAChR modeled on the Torpedo structure as viewed from the extracellular side in a slab-representation of the transmembrane domain. Subunits are labeled with greek letters and transmembrane segments M1 to M4 are indicated for one of the two α-subunits. The 12’ M2 residue, T254, is in purple stick representation.

Fig 2

Relative Orientation of the M2 and M3 transmembrane segments of the muscle nAChR αsubunit. (a) Orientation of the muscle nAChR homology modelM278 based on the Torpedo nAChR structure (PDB# 2BG9), and (b) based on the Caenorhabditis elegans glutamate-gated chloride channel (GluCl) α-subunit structure (PDB# 3RHW) as a template. Residue M2 αT254 is colored purple. M3 residues, which are selected and replaced to Cys according to the GluCl structure are color-coded as follows: Y277, pink; M278, green; L279, yellow; F280, cyan; T281, orange; M282, dark blue; V283, grey. Side chains in red color are shown for the M3 residues S288 to V293, which are in close proximity to M2 αT254 based on the Torpedo structure. (c) and (d) Same as in (a) and (b) except that only M2 and M3 are shown for clarity in stick representation. Note that in (a) and (c)αT254C is in close proximity to the red-colored side-chains and in (b) and (d) it is close to the differently-colored side-chains.

Fig 3

Concentration response curves for oocytes expressing wild-type and mutant mouse muscle nACh receptors. (a) Data for wild-type (wt) and the mutant with the lowest and highest EC₅₀ for the GluCl set are shown. (b) Data for wild-type and the mutants with the lowest and highest EC₅₀ for the Torpedo set are shown. Currents were normalized to the ACh maximum response for individual oocytes. The data were fit to a sigmoidal dose-response curve shown by the solid line. Points represent the averages (±SEM) from at least 3 oocytes.

Fig 4

Current traces for effect of oxidation and reduction. Representative ACh-induced current traces recorded before and after DTT (10 mM, 2 min), Cu:Phen (100:200 uM, 2 min) and EGTA (2mM, 2 min) applications for wild-type and mutant receptors. Current traces during reagent application are not shown. The bars above the traces indicate the duration of application of ACh; the arrows indicate the application of the given reagent. Holding potential was −60 mV.

Fig 5

DTT and Cu:Phen effects on ACh-induced current amplitudes. (a) Effect of DTT (10 mM, 2 min) and (b) Cu:Phen (100:200 μM, 2 min) on ACh-induced EC_20–50 current amplitudes for wild-type (wt), single and double Cys mutants. GluCl-based and Torpedo-based mutants are indicated in panel A. Holding potential was −60 mV. Percentage ACh-induced current amplitude after DTT or Cu:Phen treatment is shown. One-way ANOVA with Dunnett's post-test vs. wild-type shown as *** for p < 0.0001. (c) and (d) For the double mutants with significant effect of Cu:Phen application as shown in (b) the reversal of this effect was investigated by recording the effect of application of EGTA (2 mM, 2 min), and DTT (10 mM, 2 min) on current amplitudes. One-way ANOVA with Dunnett's post-test vs. the initial DTT application shown as ** for p < 0.05, and *** for p < 0.001.