THE BINDING OF PROPRANOLOL AT 5-HYDROXYTRYPTAMINE(1D-BETA) T355N MUTANT RECEPTORS MAY INVOLVE FORMATION OF 2 HYDROGEN-BONDS TO ASPARAGINE

Although the beta-adrenergic receptor antagonist (-)-propranolol binds with relatively low affinity at human 5-hydroxytryptamine(1D beta) re ceptors (K-i = 10,200 nM), it displays significantly higher affinity ( K-i = 17 nM) at its species homolog, 5-HT1B receptors, and at a mutant 5-HT1D beta receptor (K-i = 16 nM), where the threonine residue at po sition 355 (T355) is replaced with an asparagine residue (i.e., a T355 N mutant). Propranolol contains two oxygen atoms, an ether oxygen atom and a hydroxyl oxygen atom, and it has been speculated that the enhan ced affinity of propranolol for the T355N mutant receptor is related t o the ability of the asparagine residue to hydrogen bond with the ethe r oxygen atom. However, the specific involvement of the propranolol ox ygen atoms in binding to the wild-type and T355N mutant 5-HT1D beta re ceptors has never been addressed experimentally. A modification of a p reviously described 5-HT1D beta receptor graphic model was mutated by replacement of T355 with asparagine. Propranolol was docked with the w ild-type and T355N mutant 5-HT1D beta receptor models in an attempt to understand the difference in affinity of the ligand for the receptors . The binding models suggest that the asparagine residue of the mutant receptor can form hydrogen bonds with both oxygen atoms of propranolo l, whereas the threonine moiety of the wild-type receptor can hydrogen -bond only to one oxygen atom. To test this hypothesis, we prepared an d examined several analogues of propranolol that lacked either one or both oxygen atoms. The results of radioligand binding experiments are consistent with the hypothesis that both oxygen atoms of propranolol c ould participate in binding to the mutant receptor, whereas only the e ther oxygen atom participates in binding to the wild-type receptor. As such, this is the first investigation of serotonin receptors that com bines the use of molecular modeling, mutant receptors generated by sit e-directed mutagenesis, and synthesis to investigate structure/affinit y relationships.