Interaction of the α7-nicotinic subunit with its human-specific duplicated dupα7 isoform in mammalian cells: Relevance in human inflammatory responses

The 7 nicotinic receptor subunit and its partially duplicated human-specific dup7 isoform are coexpressed in neuronal and non-neuronal cells. In these cells, 7 subunits form homopentameric 7 nicotinic acetylcholine receptors (7-nAChRs) implicated in numerous pathologies. In immune cells, 7-nAChRs are essential for vagal control of inflammatory response in sepsis. Recent studies show that the dup7 subunit is a dominant-negative regulator of 7-nAChR activity in Xenopus oocytes. However, its biological significance in mammalian cells, particularly immune cells, remains unexplored, as the duplicated form is indistinguishable from the original subunit in standard tests. Here, using immunocytochemistry, confocal microscopy, coimmunoprecipitation, FRET, flow cytometry, and ELISA, we addressed this challenge in GH4C1 rat pituitary cells and RAW264.7 murine macrophages transfected with epitope- and fluorescent protein-tagged 7 or dup7. We used quantitative RT-PCR of dup7 gene expression levels in peripheral blood mononuclear cells (PBMCs) from patients with sepsis to analyze its relationship with PBMC 7 mRNA levels and with serum concentrations of inflammatory markers. We found that a physical interaction between dup7 and 7 subunits in both cell lines generates heteromeric nAChRs that remain mainly trapped in the endoplasmic reticulum. The dup7 sequestration of 7 subunits reduced membrane expression of functional 7-nAChRs, attenuating their anti-inflammatory capacity in lipopolysaccharide-stimulated macrophages. Moreover, the PBMC's dup7 levels correlated inversely with their 7 levels and directly with the magnitude of the patients' inflammatory state. These results indicate that dup7 probably reduces human vagal anti-inflammatory responses and suggest its involvement in other 7-nAChR-mediated pathophysiological processes.