Identifying Proteins of the Murine α7‐Nicotinic Acetylcholine Receptor Interactome

Abstract

The α7-Nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated channel widely expressed in vertebrates, and comprises the principal α-bungarotoxin (α-bgtx) binding protein in the mammalian central nervous system. The proteins that either interact with the α7-nAChR or are associated with it in a protein complex can be referred to as the α7-nAChR interactome. To identify the α7-nAChR interactome in neural tissue, α-bgtx-sensitive protein complexes were isolated from mouse whole brain homogenates using α-bgtx affinity beads. Proteins were eluted, reduced and alkylated before being digested with trypsin in-solution and resolved with an Orbitrap Fusion mass spectrometer at Harvard Medical School. We used the ProteoIQ™ software suite for further analysis. Identified proteins from samples isolated using α-bgtx-affinity beads and those isolated on beads lacking α-bgtx were compared. Only proteins unique to the α-bgtx affinity beads isolation were considered to be α7-nAChR associated proteins. A total of 171 candidate associated proteins were identified (i.e., with 1% FDR, >90% probability of correct assignment, identified in 2 or more replicates, 0% probability of identification in controls). These results are a continuation of our investigation of α7-nAChR interacting proteins using the latest technologies in mass spectrometry. The interactome identified here includes proteins which may affect α7-nAChR expression, localization, or modulation. Further study of these proteins may contribute to a better understanding of how they affect α7-nAChR function and the role they may have in human disease. This research was supported by NIH 1R21AG038774, 1S10RR027027, NSF EPS-1004057.