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

Nelli Mnatsakanyan, Michaela Jansen

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

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. The vertical alignment between the channel-lining transmembrane segment M2 and segment M3 in Cys-loop ligand-gated ion channels is a topic of controversy. While the vertical alignments as observed in X-ray structures of prokaryotic Gloeobacter violaceus ligand-gated ion channel (GLIC) and eukaryotic Caenorhabditis elegans glutamate-gated chloride channel α (GluClα) are in agreement, the Torpedo nAChR model based on electronmicroscopy differs. We determined the register between M2 and M3 in muscle nAChR by disulfide crosslinking. Our results are best reflected if modeled on GluClα, but not if modeled on the current Torpedo nAChR model. Overall, this indicates that the vertical alignment in nAChR is conserved between prokaryotes and eukaryotes.

Original languageEnglish (US)
Pages (from-to)843-854
Number of pages12
JournalJournal of neurochemistry
Volume125
Issue number6
DOIs
StatePublished - Jun 2013

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cellular and Molecular Neuroscience

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