Molecular insights into the α6β4 nicotinic acetylcholine receptor function and ligand recognition

The α6β4 nicotinic acetylcholine receptor (nAChR) is found in the sensory neurons of dorsal root ganglia. It is a promising therapeutic target for pain. However, the difficultly of heterologous functional expression of α6β4 receptor has hindered the discovery of drugs that target it. Here, we functionally express the human α6β4 receptor and determine the cryo-EM structures of α6β4 receptor in complex with its agonists, nicotine and the preclinical drug tebanicline. These structures were captured in non-conducting desensitized states. We elucidate that the stoichiometry of α- and β- subunits in the α6β4 receptor is 2α6:3β4. Furthermore, we identify the binding pockets for nicotine and tebanicline, demonstrating the essential residues contributing to ligand affinity and providing detailed molecular insights into why these agonists have different binding affinities despite both occupying the orthosteric site of the α6β4 receptor. These structures offer significant molecular insight into the function and ligand recognition of α6β4 receptor.