The 5-hydroxytryptamine type-3 receptor (5-HT 3 R) is a pentameric ligand-gated ion channel activated by the binding of the neurotransmitter serotonin (5-HT). This receptor plays important roles in gut-brain circuitry and is known for regulating both gut motility and emetic response. Extensive release of serotonin by enterochromaffin cells in the gastrointestinal tract can lead to 5-HT 3 R hyperactivity and dysregulation of gut-brain signaling pathways. This dysregulation has been linked to several gastrointestinal conditions, such as irritable bowel syndrome and chemotherapy-induced nausea and vomiting. 5-HT 3 Rs have additionally been implicated in psychiatric disorders, such as obsessive compulsive disorder, anxiety, and depression, making it a complex therapeutic target and underscoring a need for deeper understanding of its modulation. Cryo-EM structures have been solved with focus on the receptor’s modulation by orthosteric agonists, antagonists, and most recently partial agonists. While studying structures of this receptor bound to orthosteric ligands provides information critical for therapeutic development, a comprehensive understanding of its allosteric modulation remains limited. In this study, we investigate the binding and effects of the potent 5-HT 3 R positive allosteric modulator, 5-chloroindole, on orthosteric ligand efficacy. We will provide cryo-EM structures of the homopentameric 5-HT 3A R bound to orthosteric ligands and 5-chloroindole, as well as experiments utilizing both site-directed mutagenesis and the two-electrode voltage-clamp technique to validate ligand binding sites and function. These cryo-EM structures and electrophysiological studies both build on our current understanding of orthosteric ligand efficacy and propose mechanisms of action for 5-HT 3 R allosteric modulators.
Stauffer et al. (2026) studied this question.