An accessory orthosteric ligand binding site in the (α3)3(β4)2 nicotinic acetylcholine receptor regulates channel activation

The α3β4 nicotinic acetylcholine receptor (nAChR) is a heteropentameric ligand-gated channel whose distribution includes reward circuits of the brain, where it regulates expression of drug withdrawal symptoms. α3β4 nAChRs form two stoichiometries in the absence of other subunits, containing orthosteric binding sites between the primary α3 and the adjacent β4 subunit. The identity of the fifth (accessory) subunit is different between the two stoichiometries, but it is unknown whether this structural nuance yields any biophysical differences. To investigate this, we constrained receptor stoichiometry by engineering an α3β4 tandem dimer and co-expressing it in HEK293T cells with a select fifth subunit. We then used substituted-cysteine accessibility to covalently modify the primary and accessory binding sites. This approach revealed a functional "accessory orthosteric" site at the α3-α3 interface of (α3)₃(β4)₂ nAChRs. Understanding the stoichiometric differences in α3β4 nAChRs will inform future therapeutic strategies to target nicotine withdrawal.