Resistance to Inhibitors of Cholinesterase 3 (Ric-3) Expression Promotes Selective Protein Associations with the Human α7-Nicotinic Acetylcholine Receptor Interactome

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.

NS6740, an α7 nicotinic acetylcholine receptor silent agonist, disrupts hippocampal synaptic plasticity

“…The activity of GTS-21, NS6740, other α7 silent agonists, and allosteric modulators [1,15,17,32,34,56] in models of inflammatory disease and neuropathic pain appear to be independent of ion channel activation and, rather, depend on activation and modulation of intracellular signal transduction pathways [4,14,26,49,54]. In the case of the α7-mediated control of CAP, the cellular mediators of activity are themselves not even competent for generating nAChR channel currents, and so we must consider the complete receptor protein [50], multiple conformational states, and the complete receptor interactome [29,42] as mediators of the activity.…”

Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

“…Previous publications report difficulties showing functional α7nAChR ion channels in macrophages or similar cell types (reviewed in [19] but see [25]). As such, it has been proposed that these cells express metabotropic rather than ionotropic α7nAChRs [26]. We also tested other cell lines ( Figure S1A) and found that SH-SY5Y, not SH-EP1, cells express NACHO.…”

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

“…The same peptide was identified previously in SH-SY5Y samples (not shown) and in transfected SH-EP1 cells, two human neuroblastoma-derived cell models of α 7-nAChR expression. 34 In addition to identifying α 7-nAChRs, GABA A R subunits, recently shown to bind α -bgtx, were identified in both α -bgtx and non- α 7-nAChR, α -bgtx interactomes, but not in the α 7-nAChR interactome (Table 1). 21,35 This observation stresses the importance of controls that are α 7-nAChR-specific when using α -bgtx-affnity immobilization.…”