The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT 3 R) are members of the ligand-gated ion channel gene family. Both receptors are inhibited by nanomolar concentrations of d-tubocurarine (curare) in a competitive fashion. Chemical labeling studies on the AChR have identified tryptophan residues on the ␥ (␥Trp-55) and ␦ (␦Trp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT 3 R shows that a tryptophan residue is found in the homologous position in the 5HT 3 R (Trp-89), suggesting that this residue may be involved in curare-5HT 3 R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT 3 R for the antagonists curare and granisetron but has little effect on the affinity for the agonist serotonin. To further examine the role of this region of the receptor in ligand-receptor interactions, alanine-scanning mutagenesis analysis of the region centered on Trp-89 (Thr-85 to Trp-94) was carried out, and the ligand binding properties of the mutant receptors were determined. Within this region of the receptor, curare affinity is reduced by substitution only at Trp-89, whereas serotonin affinity is reduced only by substitution at Arg-91. On the other hand, granisetron affinity is reduced by substitutions at Trp-89, Arg-91, and Tyr-93. This differential effect of substitutions on ligand affinity suggests that different ligands may have different points of interaction within the ligand-binding pocket. In addition, the every-other-residue periodicity of the effects on granisetron affinity strongly suggests that this region of the ligand-binding site of the 5HT 3 R (and by inference, other members of the ligand-gated ion channel family) is in a ␤-strand conformation.The serotonin type 3 receptor (5HT 3 R) 1 is a member of a superfamily of ligand-gated ion channels, which includes the muscle and neuronal nicotinic acetylcholine receptor (AChR), the glycine receptor, and the ␥-aminobutyric acid type A receptor (1-3). Like the other members of the gene superfamily, the 5HT 3 R exhibits a large degree of sequence similarity, and thus presumably structural homology, with the AChR (4). Chimeras containing the amino-terminal domain of the ␣7 neuronal AChR and the carboxyl-terminal domain of the 5HT 3 R form functional ligand-gated channels with ligand specificities characteristic of AChR and permeability properties of 5HT 3 Rs (5). These experiments suggest that the AChR and 5HT 3 R have quite similar structures and signal transduction mechanisms.A large number of studies have been conducted to elucidate the structure of the ligand-binding site for AChRs (6, 7); however, very little is known about the interactions between ligands and the 5HT 3 R. A common competitive antagonist of the AChR and 5HT 3 R is d-tubocurarine (curare), which inhibits both receptors at nanomolar concentrations (8, 9). If both receptors are similar in structure, it seems reasonable to assume that similar regions of the receptors may...