RIC-3 Interacts Directly with the 5-HT _3A Receptor to Mediate Trafficking Across Subcellular Compartments

The serotonin type 3A (5-HT_3A) receptor is a pentameric ligand-gated ion channel (pLGIC) in central and peripheral neurons that conducts sodium and potassium ions upon serotonin binding. 5-HT3 receptors modulate neurotransmission and synaptic plasticity, influencing mood, sleep, appetite, and addiction. Disruptions in serotonin signaling are linked to central nervous system disorders, including schizophrenia, anxiety and depression. Clinically, these receptors are targeted by antagonists to treat chemotherapy-induced nausea and vomiting. The functional surface expression of these channels is regulated by the chaperone protein Resistant to Inhibitors of Cholinesterase 3 (RIC-3) that promotes plasma-membrane expression, maturation, and trafficking of 5-HT_3A and nicotinic acetylcholine receptors. Our previous work identified a duplicated RIC-3 binding motif within the 5-HT_3A intracellular domain (ICD). However, it was unclear whether this interaction reflected native conditions. Here, we used a recombinant 5-HT_3A ICD peptide in peptide-resin pull-down assays to investigate RIC-3 Interactions in plasma membrane (PM) fractions from <i>Xenopus</i> oocytes, endoplasmic reticulum (ER) fractions from SH-SY5Y cells, and mouse brain tissue. Across all tested systems, the 5-HT_3A ICD peptide specifically bound RIC-3. Furthermore, RIC-3 knockdown (RIC-3 KD) SH-SY5Y cells showed a marked reduction in peptide binding and decreased surface levels of nAChRα7 and 5-HT_3A receptors. These results demonstrate RIC-3-5-HT_3A ICD interaction in native cellular contexts and support a role for RIC-3 in regulating receptor surface expression and neuronal signaling.