Discovery of\nAntinociceptive α9α10 Nicotinic\nAcetylcholine Receptor Antagonists by Stable Receptor Expression

Chronic neuropathic pain is an increasingly prevalent\nsocietal\nissue that responds poorly to existing therapeutic strategies. The\nα9α10 nicotinic acetylcholine receptor (nAChR) has emerged\nas a potential target to treat neuropathic pain. However, challenges\nin expressing functional α9α10 nAChRs in mammalian cell\nlines have slowed the discovery of α9α10 ligands and studies\ninto the relationship between α9α10 nAChRs and neuropathic\npain. Here, we develop a cell line in the HEK293 background that stably\nexpresses functional α9α10 nAChRs. By also developing\ncell lines expressing only α9 and α10 subunits, we identify\ndistinct receptor pharmacology between homomeric α9 or α10\nand heteromeric α9α10 nAChRs. Moreover, we demonstrate\nthat incubation with nAChR ligands differentially regulates the expression\nof α9- or α10-containing nAChRs, suggesting a possible\nmechanism by which ligands may modify receptor composition and trafficking\nin α9- and α10-expressing cells. We then apply our α9α10\ncell line in a screen of FDA-approved and investigational drugs to\nidentify α9α10 ligands that provide new tools to probe\nα9α10 nAChR function. We demonstrate that one compound\nfrom this screen, diphenidol, possesses antinociceptive activity in\na murine model of neuropathic pain. These results expand our understanding\nof α9α10 receptor pharmacology and provide new starting\npoints for developing efficacious neuropathic pain treatments.