The Role of Tryptophan Residues in the 5-Hydroxytryptamine3 Receptor Ligand Binding Domain

Avron D Spier,Sarah C.R Lummis

doi:10.1074/jbc.275.8.5620

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.

Highlights

The 5-HT31 receptor is a member of the Cys loop family of ligand-gated ion channels, which includes nicotinic acetylcholine, ␥-aminobutyric acid (GABA), and glycine receptors
NACh receptors indicate that the ligand binding site is located in discontiguous regions of the extracellular N-terminal domain, and this has been further confirmed by the construction of a chimeric protein consisting of the N-terminal domain of the ␣7 neuronal nicotinic acetylcholine (nACh) receptor subunit linked to the C-terminal portion of the 5-HT3A receptor subunit, which showed nACh receptor pharmacological properties and 5-HT3 receptor channel properties [11]
Labeling and mutagenesis studies have identified a number of N-terminal amino acids in nACh subunits that are probably involved in ligand binding; these are mostly aromatic amino acids and include Trp␣54, Trp␣86, Tyr␣93, Trp␣149, Trp␣187, Tyr␣190, Cys␣192, Cys␣193, and Tyr␣198 [12,13,14,15,16,17,18,19,20,21,22,23,24,25]

Summary

Introduction

The 5-HT31 receptor is a member of the Cys loop family of ligand-gated ion channels, which includes nicotinic acetylcholine (nACh), ␥-aminobutyric acid (GABA), and glycine receptors. The predicted 5-HT3 receptor subunit structure shows the expected characteristics of a nACh receptor-type subunit, including a large extracellular N-terminal region, and four putative transmembrane domains (TM1–TM4) with a bulky intracellular loop between TM3 and TM4. Labeling and mutagenesis studies have identified a number of N-terminal amino acids in nACh subunits that are probably involved in ligand binding; these are mostly aromatic amino acids and include Trp␣54, Trp␣86, Tyr␣93, Trp␣149, Trp␣187, Tyr␣190, Cys␣192, Cys␣193, and Tyr␣198 [12,13,14,15,16,17,18,19,20,21,22,23,24,25]

Results

Discussion

Conclusion