Duplicated binding site for RIC-3 in 5-Hydroxytryptamine Receptors Subtype 3

Abstract

Serotonin or 5-hydroxytryptamine receptors type 3 (5-HT3) belong to the family of pentameric ligand-gated ion channels (pLGICs), which also includes other neurotransmitter-gated ion channels such as nicotinic acetylcholine receptors, γ-amino butyric acid A receptors, and glycine receptors. pLGICs have been long-standing clinical targets for treating psychiatric disorders such as anxiety, schizophrenia, addiction, and neurological diseases like Alzheimers and Parkinsons disease. Due to the highly-homologous structures of pLGICs extracellular and transmembrane domains, clinical trials for drug candidates targeting these domains have been largely hampered by undesired effects mediated by off-subunit modulation. With the present study, we explore the interaction interface of the 5-HT3A intracellular domain (ICD) with the resistance to inhibitors of choline esterase (RIC-3) chaperone. Protein-protein interactions are attractive and successful targets for contemporary drug development. Previously, we have shown that RIC-3 physically interacts with 5-HT3A subunits, and we identified the L1-MX fragment of the ICD fused to maltose-binding protein as sufficient for interaction. For the present study, synthetic L1-MX-based fragments and Ala-scanning were used to probe binding to RIC-3 using a pull-down assay. In complementary studies with full-length 5-HT3A subunits, we investigated the impact of these substitutions on modulation of functional surface expression by RIC-3 using two-electrode voltage-clamp recording after expression in Xenopus laevis oocytes. Our data demonstrate that the 5-HT3A-MX segment contains the interaction interface, and that residues W347, R349, and L353 are critical for the interaction with RIC-3.