Ondansetron and Granisetron Binding Orientation in the 5‑HT_3 Receptor Determined by Unnatural Amino Acid Mutagenesis

The serotonin type 3 receptor (5-HT_3R) is a ligand-gated ion channel found in the central and peripheral nervous systems. The 5-HT_3R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT_3A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket.