BPS2026 – Exploring heteromeric 5-HT3 receptor pairings using FRET and FIR

5-HT 3 receptors are pentameric, ligand-gated ion channels (pLGICs) distributed across the peripheral and central nervous systems. There are five known subunits that can form 5-HT 3 receptors: 5-HT 3A-E . 5-HT 3A can assemble into functional homomeric receptors, whereas 5-HT 3B-E are only functional in heteromeric pairings with 5-HT 3A . Heteromeric pairings between 5-HT 3A and 5-HT 3B produce receptors with altered biophysical properties and an A 3 B 2 subunit stoichiometry, as demonstrated by Forster resonance energy transfer (FRET) and fluorescence intensity ratio (FIR) experiments. Comparatively little is known about heteromeric pairings between 5-HT 3A and 5-HT 3C-E . We synthesized the genes for human 5-HT 3A-E receptors and inserted cyan or yellow fluorescent protein tags (CFP or YFP) in the unstructured loop between transmembrane domains 3 and 4. Using a spectral FRET approach, we measured the interaction between tagged 5-HT 3B-E and 5-HT 3A in HEK293T cells. From these results, we verified that 5-HT 3A co-assembles with 5-HT 3B and also assembles with 5-HT 3C and 5-HT 3D . FIR measurements confirmed the A 3 B 2 stoichiometry of 5-HT 3A /5-HT 3B pairings but, surprisingly, were most consistent with a A 4 X 1 stoichiometry for 5-HT 3A /5-HT 3C and 5-HT 3A /5-HT 3D receptors.