BPS2026 – Direct interaction of RIC3 with 5HT3A receptors in mammalian cells

5-HT3A receptor is a distinctive member of the serotonin receptor family, expressed in central and peripheral nervous system neurons. Unlike most serotonin receptors, which are G-protein-coupled, 5-HT3A belongs to the pentameric ligand-gated ion channel family. Upon serotonin binding, it forms a cation-selective pore that allows sodium and potassium flux, shaping excitatory neurotransmission. Through this activity, 5-HT3 receptors contribute to synaptic plasticity and physiological processes, including mood, emotion, sleep, appetite, and addiction. Aberrant serotonin signaling, and 5-HT3 receptor dysfunction, has been linked to neuropsychiatric disorders such as schizophrenia, anxiety, and depression. Clinically, antagonists of the 5-HT3 receptor are well established in the management of chemotherapy-induced nausea and vomiting, underscoring the receptor’s therapeutic relevance. The functional expression of 5-HT3A at the neuronal surface requires precise quality control within the endoplasmic reticulum. A central regulator of this process is RIC-3, an ER-resident chaperone protein that promotes receptor folding, maturation, and forward trafficking to the plasma membrane. Through this role, RIC-3 indirectly modulates neuronal excitability and synaptic communication. Earlier, we identified a direct interaction between RIC-3 and the intracellular domain (ICD) of 5-HT3A, mediated by a duplicated RIC-3 binding motif within the ICD. Yet, the physiological relevance of this interaction in mammalian cells and its precise subcellular context remained unresolved. In the present study, we investigated this mechanism using peptide-resin pull-down assays with a recombinant 5-HT3A ICD peptide. Analyses of plasma membrane fractions from Xenopus laevis oocytes, together with ER fractions from SH-SY5Y neuroblastoma cells and mouse brain tissue, demonstrated binding of the ICD peptide to RIC-3. Furthermore, RIC-3 knockdown in SH-SY5Y cells markedly reduced peptide binding as well as α7 and 5-HT3A surface expression. These findings establish direct interaction between RIC-3 and 5-HT3A, extending prior observations into mammalian systems and reinforcing its physiological significance.