Characterization of the ligand-binding site of the serotonin 5-HT3 receptor: the role of glutamate residues 97, 224, AND 235.

Détails

ID Serval
serval:BIB_61F1EF051D0B
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Characterization of the ligand-binding site of the serotonin 5-HT3 receptor: the role of glutamate residues 97, 224, AND 235.
Périodique
Journal of Biological Chemistry
Auteur(s)
Schreiter C., Hovius R., Costioli M., Pick H., Kellenberger S., Schild L., Vogel H.
ISSN
0021-9258[print], 0021-9258[linking]
Statut éditorial
Publié
Date de publication
2003
Volume
278
Numéro
25
Pages
22709-22716
Langue
anglais
Résumé
Ligand-gated ion channels of the Cys loop family are receptors for small amine-containing neurotransmitters. Charged amino acids are strongly conserved in the ligand-binding domain of these receptor proteins. To investigate the role of particular residues in ligand binding of the serotonin 5-HT3AS receptor (5-HT3R), glutamate amino acid residues at three different positions, Glu97, Glu224, and Glu235, in the extracellular N-terminal domain were substituted with aspartate and glutamine using site-directed mutagenesis. Wild type and mutant receptor proteins were expressed in HEK293 cells and analyzed by electrophysiology, radioligand binding, fluorescence measurements, and immunochemistry. A structural model of the ligand-binding domain of the 5-HT3R based on the acetylcholine binding protein revealed the position of the mutated amino acids. Our results demonstrate that mutations of Glu97, distant from the ligand-binding site, had little effect on the receptor, whereas mutations Glu224 and Glu235, close to the predicted binding site, are indeed important for ligand binding. Mutations E224Q, E224D, and E235Q decreased EC50 and Kd values 5-20-fold, whereas E235D was functionally expressed at a low level and had a more than 100-fold increased EC50 value. Comparison of the fluorescence properties of a fluorescein-labeled antagonist upon binding to wild type 5-HT3R and E235Q, allowed us to localize Glu235 within a distance of 1 nm around the ligand-binding site, as proposed by our model.
Mots-clé
Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, Cell Line, Glutamic Acid, Granisetron/pharmacology, Humans, Ligands, Membrane Potentials/drug effects, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Patch-Clamp Techniques, Protein Subunits/chemistry, Protein Subunits/genetics, Radioligand Assay, Receptors, Serotonin/chemistry, Receptors, Serotonin/genetics, Receptors, Serotonin, 5-HT3, Sequence Alignment, Sequence Homology, Amino Acid, Transfection, Tritium
Pubmed
Web of science
Open Access
Oui
Création de la notice
24/01/2008 13:45
Dernière modification de la notice
20/08/2019 15:18
Données d'usage