Abstract

The agonist binding sites of the fetal muscle nicotinic acetylcholine receptor are formed at the interfaces of α-subunits and neighboring γ- and δ-subunits. When the receptor is in the nonconducting desensitized state, the α-γ site binds the agonist epibatidine 200-fold more tightly than does the α-δ site. To determine the structural basis for this selectivity, we constructed γ/δ-subunit chimeras, coexpressed them with complementary wild-type subunits in HEK 293 cells, and determined epibatidine affinity of the resulting complexes. The results reveal three determinants of epibatidine selectivity: γ104–117/δ106–δ119, γ164–171/δ166–177, and γPro190/δAla196. Point mutations reveal that three sequence differences within the γ104–117/δ106–δ119 region are determinants of epibatidine selectivity: γLys104/δTyr106, γSer111/δTyr113, and γTyr117/δTyr119. In the δ-subunit, simultaneous mutation of these residues to their γ equivalent produces high affinity, γ-like epibatidine binding. However, converting γ to δ affinity requires replacement of the γ104–117 segment with δ sequence, suggesting interplay of residues in this region. The structural basis for epibatidine selectivity is explained by computational docking of epibatidine to a homology model of the α-γ binding site.