Characterisation of heteromeric 5-HT₃ receptors: a focus on the 5-HT₃C and 5-HT₃E subunits

5-HT₃ receptors are ligand-gated cation channels present in both central and peripheral nervous systems. 5-HT₃ receptor antagonists are used clinically for a range of disorders including irritable bowel syndrome, chemotherapy-induced nausea and depression but have varying efficacy. 5-HT₃ receptors potentially contain five different subunits (A, B, C, D and E) that may contribute to the variability in efficacy. Inter-species variations in expression pattern of the subunit have also been reported including in human and rodents. The present study was aimed to characterise the expression, functional and pharmacological profiles of human 5-HT₃ receptors containing C and/or E subunits. Quantitative real-time PCR of human colon and ileum tissue was used to determine mRNA expression for all 5-HT₃ receptor subunits. Development of cloning strategies to optimize expression of heteromeric 5-HT₃ receptors in HEK293T cells preceded the functional and pharmacological experiments using whole cell patch clamping. The overall expression pattern of the C subunit did not vary significantly from A and B subunits in both human colon and ileum layers and regions. However the E subunit was found predominantly in mucosa of colon and ileum but not muscle layers. Only subtle changes were seen in the electrical properties of cells expressing 5-HT₃A, 5-HT₃AC, 5-HT₃AE, or 5-HT₃ACE subunits shown by whole cell recordings. The tri-heteromeric 5-HT₃ACE receptor was significantly inhibited by ondansetron and palonosetron. Most notably, palonosetron exerted faster inhibition on tri-heteromeric 5-HT₃ACE receptor compared to its homomeric and di-heteromeric counterparts. The mutant 5-HT₃A+C[N163K] receptor produced a significantly larger peak amplitude in response to 30µM 5-HT compared to the wild-type, but no significant difference on the relative response to ondansetron and palonosetron were observed. The widespread distribution of the C and E subunits throughout the human colon suggest that they may contribute to physiological functions. The predominant location of the E subunit in the mucosa could make it a potential therapeutic target. Patch-clamp experiments indicate that C and/or E subunits in heteromeric receptors may alter efficacies of 5-HT and clinically used antagonists such as palonosetron, which deserve further exploration to gain greater insights to improve current therapeutic applications of 5-HT₃ receptor antagonists including personalized therapies.