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  4. Characterization of the Ligand-binding Site of the Serotonin 5-HT3 Receptor: The Role of Glutamate Residues 97, 224,and 235
 
research article

Characterization of the Ligand-binding Site of the Serotonin 5-HT3 Receptor: The Role of Glutamate Residues 97, 224,and 235

Schreiter, Christoph  
•
Hovius, Ruud  
•
Costioli, Matteo
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2003
Journal of Biological Chemistry

Ligand-gated ion channels of the Cys loop family are receptors for small amine-contg. 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 electrophysiol., radioligand binding, fluorescence measurements, and immunochem. 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. [on SciFinder (R)]

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Type
research article
DOI
10.1074/jbc.M301801200
Web of Science ID

WOS:000183503900072

Author(s)
Schreiter, Christoph  
Hovius, Ruud  
Costioli, Matteo
Pick, Horst  
Kellenberger, Stephan
Schild, Laurent
Vogel, Horst  
Date Issued

2003

Published in
Journal of Biological Chemistry
Volume

278

Issue

25

Start page

22709

End page

22716

Editorial or Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
LCPPM  
Available on Infoscience
February 27, 2006
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/226379
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