The role of tryptophan residues in the 5-Hydroxytryptamine(3) receptor ligand binding domain

Abstract

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 tryptophan residues in the ligand binding domain of the 5-hydroxytryptamine(3) (5-HT(3)) receptor, we used site-directed mutagenesis to change each of the eight N-terminal tryptophan residues in the 5-HT(3A) receptor subunit to tyrosine or serine. The mutants were expressed as homomeric 5-HT(3A) receptors in HEK293 cells and analyzed with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Trp(90), Trp(183), and Trp(195) to tyrosine resulted in functional receptors, although with increased EC(50) values (2-92-fold) to 5-HT(3) receptor agonists. Changing these residues to serine either ablated function (Trp(90) and Trp(183)) or resulted in a further increase in EC(50) (Trp(195)). Mutation of residue Trp(60) had no effect on ligand binding or receptor function, whereas mutation of Trp(95), Trp(102), Trp(121), and Trp(214) ablated ligand binding and receptor function, and all but one of the receptors containing these mutations were not expressed at the plasma membrane. We propose that Trp(90), Trp(183), and Trp(195) are intimately involved in ligand binding, whereas Trp(95), Trp(102), Trp(121), and Trp(214) have a critical role in receptor structure or assembly.