Putative α7-selective ligands interact with α9-containing nicotinic acetylcholine receptors and modulate immune...

Nicotinic acetylcholine receptors (nAChRs) on immune cells are promising therapeutic targets for the treatment of inflammatory diseases and pain. Both α7 and α9* nAChRs (*denotes the potential presence of other nAChR subunits) have been implicated as mediators of the cholinergic anti-inflammatory system (CAS). This study investigated the binding sites of α7-selective ligands on these receptors and their effects on ATP-dependent release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 by human mononuclear phagocytes.

The effects of classical ligands (e.g. ACh, nicotine), unconventional (phosphocholine), putative α7-specific ligands (S24795, PNU-282987 and methyllycaconitine), on the ATP-induced IL-1β release were studied in lipopolysaccharide-primed human monocytic THP-1 cells and THP-1-derived macrophages. Electrophysiological two-electrode voltage-clamp measurements were conducted on Xenopus laevis oocytes expressing human α7, α9 or α9α10 nAChRs. Molecular docking was performed using the crystal structure of the homomeric human α7 receptor (PDB ID: 7EKI) and a modeled pentameric assembly of the homomeric α9 extracellular domain (PDB ID: 6HY7). In addition, the homomeric α10 extracellular domain was generated by homology modeling using 6HY7 as the template.

In cytokine-release experiments, the nAChR agonists efficiently inhibited ATP-mediated IL-1β release. This inhibitory effect was reversed by specific antagonistic conopeptides [V11L;V16D]ArIB (α7 antagonist) and RgIA4 (α9 and α9α10 antagonist), indicating the involvement of nAChRs containing subunits α7, α9 and/or α10. Electrophysiological measurements suggested an interaction of putative α7-specific ligands with human α9* nAChRs. In molecular docking simulations, all tested ligands showed reasonable binding affinity to homomeric α7, α9 and α10 nAChR models near the C-loop region of the binding pocket.

Our findings provide a more nuanced framework for interpreting the roles of nAChR subtypes in non-neuronal immune modulation, highlighting the complexity and potential importance of α9* nAChRs in the context of inflammation and innate immunity. The results underscore the importance of considering the nAChR subunit α9 when developing α7-selective ligands for immunomodulation and provides novel insights into the role of α9* nAChRs as potential therapeutic targets for inflammatory diseases and pain.