Duplicated binding site for RIC-3 chaperon protein in serotonin type 3A subunits.

Abstract

Serotonin or 5-hydroxytryptamine type 3A receptors (5-HT3A) belong to the pentameric ligand-gated ion channel super-family, which have been long-standing therapeutic targets for psychiatric disorders and neurological diseases. Due to structural conservation and significant sequence similarities in the extracellular and transmembrane domains of this family, clinical trials for drug candidates targeting these two domains have been hampered by off-subunit modulation. The intracellular domain of this family, in contrast, exhibits significant diversity in length, amino acid composition, and function, and hence positions itself as a potential drug target. We therefore explored the interaction interface of the 5-HT3A intracellular domain (ICD) with its regulator, the resistance to inhibitors of choline esterase (RIC-3) protein. We have previously shown that RIC-3 interacts with the L1-MX segment of the ICD fused to maltose-binding protein. In this study, using synthetic L1-MX-based peptides, Ala-scanning, and a pull-down assay, we identified motif DWLRXX(X)VLDR, as a critical motif for interaction with RIC-3. This motif is repeated twice within the ICD. One site is within the MX-helix, another site is at the MAM4-helix transition. For both sites, triple-Ala substitutions (MX: W347, R349, and L353; MAM4: W447, R449, and L454) disrupt the interactions between 5-HT3A-ICD-peptide and RIC-3. Complementary studies using full-length 5-HT3A subunits confirmed that the Ala substitutions reduced the RIC-3 mediated modulation of 5-HT3A functional surface expression. We thus identified two binding sites for RIC-3 with a shared duplicated motif in 5-HT3A subunits, one in the MX-helix and one at the MAM4-helix transition.