Received for publication March 3, 2006.
Revised April 17, 2006.
Accepted for publication May 23, 2006.

Mapping residues in the ligand-binding domain of the 5-HT₃ receptor onto d-tubocurarine structure

Abstract

The serotonin 5-HT₃ receptor is a member of the cys-loop ligand-gated ion channel family. We have used the combination of site-directed mutagenesis, homology modeling of the 5-HT₃R extracellular domain, and ligand docking simulations as a way to map the architecture of the 5-HT₃R ligand binding domain. Mutation of F226 in loop C of the binding site to tyrosine (F226Y) has no effect on the apparent affinity of the competitive antagonist d-tubocurarine (dTC) for the receptor. On the other hand, replacement of N128 in loop A of the binding site with alanine (N128A) increases the apparent affinity of dTC approximately 10-fold. Double-mutant cycle analysis employing a panel of dTC analogs with substitutions at various positions to identify specific points of interactions between the dTC analogs and N128 suggests that N128 makes a direct interaction with the 2' N of dTC. Molecular modeling of the 5-HT₃R extracellular domain using the antagonist-bound conformation of the Aplysia acetylcholine binding protein (AChBP) as a template followed by ligand docking simulations produces two classes of structures of the 5-HT3₃/dTC complex, only one of which has the 2' N of dTC positioned at N128 and thus is consistent with the data from this study and previously-published data. The use of the rigid dTC analogs as "molecular rulers" in conjunction with double-mutant cycle analysis of mutant receptors can allow the spatial mapping of the position of various residues in the ligand-binding site.

Key words: Serotonin, Serotonin, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants

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