Abstract
The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel widely expressed in vertebrates and is associated with numerous physiological functions. As transmembrane ion channels, α7-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 α7-nAChRs. The putative chaperone resistance to inhibitors of cholinesterase 3 (Ric-3) has been reported to interact with, and enhance the surface expression of, α7-nAChRs. In this study, we identified proteins that associate with α7-nAChRs when Ric-3 is expressed. Using α-bungarotoxin (α-bgtx), we isolated and compared α7-nAChR-associated proteins from two stably transfected, human tumor-derived cell lines: SH-EP1-hα7 expressing human α7-nAChRs and the same cell line further transfected to express Ric-3, SH-EP1-hα7-Ric-3. Mass spectrometric analysis of peptides identified thirty-nine proteins that are associated with α7-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 α-bgtx may be used to isolate and identify α7-nAChRs as well as how the expression of chaperones such as Ric-3 can influence proteins associating with α7-nAChRs. These associating proteins may alter activities of α7-nAChRs to expand their functionally-relevant repertoire as well as to affect biogenesis and membrane trafficking of α7-nAChRs.
Highlights
The α7-nicotinic acetylcholine receptor (α7-nAChR) is a homopentameric ligand-gated ion channel widely expressed in both neuronal and non-neuronal tissue and is associated with numerous physiological processes such as memory and cognition [1]
Solubilized membrane extracts from SH-EP1-hα7-resistance to inhibitors of cholinesterase 3 (Ric-3) and SH-EP1-hα7 cell lines were incubated with α-bgtx-affinity beads to isolate α7-nAChRs for further analysis
Comparable 125I-α-bgtx binding levels were observed for α-bgtx-affinity immobilized protein from both SH-EP1-hα7-Ric-3 and SH-EP1-hα7 cell solubilized receptor preparations (56 ± 15 and 49 ± 9 respectively fmol 125I-α-bgtx/mg solubilized protein) (Fig 2)
Summary
The α7-nicotinic acetylcholine receptor (α7-nAChR) is a homopentameric ligand-gated ion channel widely expressed in both neuronal and non-neuronal tissue and is associated with numerous physiological processes such as memory and cognition [1]. Compared to other nAChR subtypes, the α7-nAChR desensitizes more rapidly, is more permeable to Ca2+, and is a target for highly selective ligands such as α-Bungarotoxin (α-bgtx), derived from the venom of the snake Bungarus multicinctus and methyllycaconitine (MLA), derived from plants of the Delphinium genus [2,3,4]. These highly selective ligands are powerful tools that enable the isolation of α7-nAChRs and associated proteins. One of the limited number of proteins previously reported to associate with α7-nAChRs, is the molecular chaperone resistance to inhibitors of cholinesterase 3 (Ric-3), which has been shown to facilitate nAChR assembly and trafficking [9, 17]
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