Pennington_2005_Mol.Pharmacol_67_123

The agonist binding sites of the fetal muscle nicotinic acetylcholine receptor are formed at the interfaces of alpha-subunits and neighboring gamma- and delta-subunits. When the receptor is in the nonconducting desensitized state, the alpha-gamma site binds the agonist epibatidine 200-fold more tightly than does the alpha-delta site. To determine the structural basis for this selectivity, we constructed gamma/delta-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: gamma104-117/delta106-delta119, gamma164-171/delta166-177, and gammaPro190/deltaAla196. Point mutations reveal that three sequence differences within the gamma104-117/delta106-delta119 region are determinants of epibatidine selectivity: gammaLys104/deltaTyr106, gammaSer111/deltaTyr113, and gammaTyr117/deltaTyr119. In the delta-subunit, simultaneous mutation of these residues to their gamma equivalent produces high affinity, gamma-like epibatidine binding. However, converting gamma to delta affinity requires replacement of the gamma104-117 segment with delta 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 alpha-gamma binding site.