The role of tryptophan residues in the 5-hydroxytryptamines receptor ligand binding domain

Aromatic amino acids are important components of the ligand binding site in the Cys loop family of ligand-gated ion channels. To examine the role of t ryptophan residues in the ligand binding domain of the 5-hydroxytryptamine( 3) (5-HT3) receptor, we used site-directed mutagenesis to change each of th e eight N-terminal tryptophan residues in the 5-HT3A receptor subunit to ty rosine or serine, The mutants were expressed as homomeric 5-HT3A receptors in HEK293 cells and analyzed with radioligand binding, electrophysiology, a nd immnunocytochemistry. Mutation of Trp(90), Trp(183), and Trp(195) to tyr osine resulted in functional receptors, although with increased EC50 values (2-92-fold) to 5-HT3 receptor agonists, Changing these residues to serine either ablated function (Trp(90) and Trp(183)) gp resulted in a further inc rease in EC50 (Trp(195)). Mutation of residue Trp(60) had no effect on Liga nd binding or receptor function, whereas mutation of Trp(95), Trp(102), Trp (121), and Trp(214) ablated ligand binding and receptor function, and all b ut one of the receptors containing these mutations were not expressed at th e plasma membrane. We propose that Trp(90), Trp(183), and Trp(195) are inti mately involved in Ligand binding, whereas Trp(95), Trp(102), Trp(121), and Trp(214) have a critical role in receptor structure or assembly.