Proteomic Investigation of Murine Neuronal alpha 7-Nicotinic Acetylcholine Receptor Interacting Proteins

摘要

The alpha 7-nicotinic acetylcholine receptor (alpha 7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity alpha-bungarotoxin (alpha-bgtx) binding protein in the mammalian CNS. alpha 7-nAChRs associate with proteins that can modulate its properties. The alpha 7-nAChR interactome is the summation of proteins interacting or associating with alpha 7-nAChRs in a protein complex. To identify an alpha 7-nAChR interactome in neural tissue, we isolated alpha-bgtx-affinity protein complexes from wild-type and alpha 7-nAChR knockout (alpha 7 KO) mouse whole brain tissue homogenates using alpha-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the alpha 7-nAChR specific ligand, alpha-bgtx, were determined to be alpha 7-nAChR associated proteins. The alpha 7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to alpha-bgtx in the absence of alpha 7-nAChR expression, thereby identifying nonspecific proteins for alpha 7-nAChR investigations using alpha-bgtx enrichment. These results expand on our previous investigations of alpha 7-nAChR interacting proteins using alpha-bgtx-affinity bead isolation by controlling for differences between alpha 7-nAChR and alpha-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The alpha 7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of alpha 7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease.