Unorthodox Acetylcholine Binding Sites Formed by α5 and β3 Accessory Subunits in α4β2*Nicotinic Acetylcholine Receptors

All nicotinic acetylcholine receptors (nAChRs) evolved from homomeric nAChRs in which all five subunits are involved in forming acetylcholine (ACh) binding sites at their interfaces. Heteromeric 42* nAChRs typically have two ACh binding sites at 4/2 interfaces and a fifth accessory subunit surrounding the central cation channel. 2 accessory subunits do not form ACh binding sites, but 4 accessory subunits do at the 4/4 interface in (42)(2)4 nAChRs. 5 and 3 are closely related subunits that had been thought to act only as accessory subunits and not take part in forming ACh binding sites. The effect of agonists at various subunit interfaces was determined by blocking homologous sites at these interfaces using the thioreactive agent 2-((trimethylammonium)ethyl) methanethiosulfonate (MTSET). We found that 5/4 and 3/4 interfaces formed ACh binding sites in (42)(2)5 and (42)(2)3 nAChRs. The 4/5 interface in (24)(2)5 nAChRs also formed an ACh binding site. Blocking of these sites with MTSET reduced the maximal ACh evoked responses of these nAChRs by 30-50%. However, site-selective agonists NS9283 (for the 4/4 site) and sazetidine-A (for the 4/2 site) did not act on the ACh sites formed by the 5/4 or 3/4 interfaces. This suggests that unorthodox sites formed by 5 and 3 subunits have unique ligand selectivity. Agonists or antagonists for these unorthodox sites might be selective and effective drugs for modulating nAChR function to treat nicotine addiction and other disorders.