Structural basis for epibatidine selectivity at desensitized nicotinic receptors.
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.
Pennington
Richard A
RA
School of Biological Sciences, University of Manchester, G38 Stopford Bldg., Oxford Rd, Manchester M13 9PT, United Kingdom.
Gao
Fan
F
Sine
Steven M
SM
Prince
Richard J
RJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D002460:Cell Line; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018722:Nicotinic Agonists / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D019086:Bridged Bicyclo Compounds, Heterocyclic; D018722:Nicotinic Agonists; D010446:Peptide Fragments; D021122:Protein Subunits; D011725:Pyridines; D011978:Receptors, Nicotinic; C082748:epibatidine