Abstract

The serotonin 5-HT₃ receptor (5-HT₃R) 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 Phe226 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 Asn128 in loop A of the binding site with alanine (N128A) increases the apparent affinity of dTC by 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 Asn128 suggests that Asn128 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 californica acetylcholine binding protein as a template followed by ligand docking simulations produces two classes of structures of the 5-HT₃R/dTC complex; only one of these has the 2′N of dTC positioned at Asn128 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.