Mulcahy_2015_PLoS.One_10_e0134409

The alpha7-nicotinic acetylcholine receptor (alpha7-nAChR) is a ligand-gated ion channel widely expressed in vertebrates and is associated with numerous physiological functions. As transmembrane ion channels, alpha7-nAChRs need to be expressed on the surface of the plasma membrane to function. The receptor has been reported to associate with proteins involved with receptor biogenesis, modulation of receptor properties, as well as intracellular signaling cascades and some of these associated proteins may affect surface expression of alpha7-nAChRs. The putative chaperone resistance to inhibitors of cholinesterase 3 (Ric-3) has been reported to interact with, and enhance the surface expression of, alpha7-nAChRs. In this study, we identified proteins that associate with alpha7-nAChRs when Ric-3 is expressed. Using alpha-bungarotoxin (alpha-bgtx), we isolated and compared alpha7-nAChR-associated proteins from two stably transfected, human tumor-derived cell lines: SH-EP1-halpha7 expressing human alpha7-nAChRs and the same cell line further transfected to express Ric-3, SH-EP1-halpha7-Ric-3. Mass spectrometric analysis of peptides identified thirty-nine proteins that are associated with alpha7-nAChRs only when Ric-3 was expressed. Significantly, and consistent with reports of Ric-3 function in the literature, several of the identified proteins are involved in biological processes that may affect nAChR surface expression such as post-translational processing of proteins, protein trafficking, and protein transport. Additionally, proteins affecting the cell cycle, the cytoskeleton, stress responses, as well as cyclic AMP- and inositol triphosphate-dependent signaling cascades were identified. These results illuminate how alpha-bgtx may be used to isolate and identify alpha7-nAChRs as well as how the expression of chaperones such as Ric-3 can influence proteins associating with alpha7-nAChRs. These associating proteins may alter activities of alpha7-nAChRs to expand their functionally-relevant repertoire as well as to affect biogenesis and membrane trafficking of alpha7-nAChRs.