Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins

The α7-nicotinic acetylcholine
receptor (α7-nAChR)
is a ligand-gated ion channel that is expressed widely in vertebrates
and is the principal high-affinity α-bungarotoxin (α-bgtx)
binding protein in the mammalian CNS. α7-nAChRs associate with
proteins that can modulate its properties. The α7-nAChR interactome
is the summation of proteins interacting or associating with α7-nAChRs
in a protein complex. To identify an α7-nAChR interactome in
neural tissue, we isolated α-bgtx-affinity protein complexes
from wild-type and α7-nAChR knockout (α7 KO) mouse whole
brain tissue homogenates using α-bgtx-affinity beads. Affinity
precipitated proteins were trypsinized and analyzed with an Orbitrap
Fusion mass spectrometer. Proteins isolated with the α7-nAChR
specific ligand, α-bgtx, were determined to be α7-nAChR
associated proteins. The α7-nAChR subunit and 120 additional
proteins were identified. Additionally, 369 proteins were identified
as binding to α-bgtx in the absence of α7-nAChR expression,
thereby identifying nonspecific proteins for α7-nAChR investigations
using α-bgtx enrichment. These results expand on our previous
investigations of α7-nAChR interacting proteins using α-bgtx-affinity
bead isolation by controlling for differences between α7-nAChR
and α-bgtx-specific proteins, developing an improved protein
isolation methodology, and incorporating the latest technology in
mass spectrometry. The α7-nAChR interactome identified in this
study includes proteins associated with the expression, localization,
function, or modulation of α7-nAChRs, and it provides a foundation
for future studies to elucidate how these interactions contribute
to human disease.