Effects of RIC-3 on the expression profile of serotonin type 3A receptors

Serotonin type 3A (5-HT3A) receptors belong to the Cys-loop ligand-gated ion channel receptor superfamily. 5-HT3A receptors are found in high densities in certain brain regions, have an involvement with brain-gut signaling circuitry and other non-serotonergic synaptic activities, which make them effective and potential therapeutic targets for treatments of many conditions such as irritable bowel syndrome, chemotherapy-induced vomiting, inflammation, and psychiatric disorders. RIC-3, a transmembrane protein and endoplasmic reticulum resident chaperone, acts as a molecular chaperone of cation-conducting Cys-loop receptors, both serotonin type 3A (5-HT3A) and nicotinic acetylcholine receptors. RIC-3 is needed for efficient receptor folding, assembly and trafficking/functional surface expression. RIC-3 interacts directly with the intracellular domain of serotonin type 3A subunits (ICD). RIC-3 has two binding sites with a shared duplicated motif in 5HT3A subunits, one in the MX-helix and one in the MAM4-helix. Toward drug discovery based on protein-protein interactions, we here aimed to determine the effect of RIC-3 on the expression profile of serotonin type 3A receptors. For this study, 5-HT3A constructs were genetically engineered, and the functional surface expression was examined using two-electrode voltage clamp and immunoblot analysis/western blot. We found that when 5-HT3A channels were co-injected with RIC-3, RIC-3 inhibits 5-HT3A current amplitudes elicited by serotonin in Xenopus oocytes; Ala replacements at residues W347, R349, and L353 (MX) or residues W447, R449, and L454 (MAM4) on the ICD weakened the inhibitory effect of RIC-3 on the functional surface expression of the channels. We infer that in Xenopus oocytes, RIC-3 reduction of functional surface expression of 5HT3A receptors is mediated by binding to the two identified binding sites.