Visualising functional 5-HT3 receptors containing A and C subunits at or near the cell surface.

Five different subunits of the human serotonin 3 (5-hydroxytrptamine 3; 5-HT3) receptor exist and these are present in both central and peripheral systems. Different subunits alter the efficacy of 5-HT3 receptor antagonists used to treat diarrhoea predominant-irritable bowel syndrome, chemotherapy induced nausea and vomiting and depression. Cell surface arrangement of 5-HT3 receptor complexes and the contribution of C, D and E subunits to receptor function is poorly understood. Here, we examine interactions of A and C subunits using 5-HT3 receptor subunits containing fluorescent protein inserts between the 3rd and 4th transmembrane spanning region. HEK293T cells that do not normally express 5-HT3 receptor subunits, were transiently transfected with A or C or both subunits. Patch clamp experiments show that cells transfected with either fluorescent protein tagged A or A and C subunits generate whole cell currents in response to 5-HT. These findings correlate with the apparent distribution of fluorescent protein tagged A and C subunits at or near cell surfaces detected using TIRF microscopy. In co-transfected cells, the A and C subunits are associated forming AC heteromer complexes at or near the cell surface and a proportion can also form A or C homomers. In conclusion, it is likely that both A homomers and AC heteromers contribute to whole cell currents in response to 5-HT with minimal contribution from C homomers.

Distribution of 5-HT3, 5-HT4, and 5-HT7 Receptors Along the Human Colon.

BACKGROUND/AIMS: Several disorders of the gastrointestinal tract are associated with abnormal serotonin (5-HT) signaling or metabolism where the 5-HT3 and 5-HT4 receptors are clinically relevant. The aim was to examine the distribution of 5-HT3, 5-HT4, and 5-HT7 receptors in the normal human colon and how this is associated with receptor interacting chaperone 3, G protein coupled receptor kin-ases, and protein LIN-7 homologs to extend previous observations limited to the sigmoid colon or the upper intestine. METHODS: Samples from ascending, transverse, descending, and sigmoid human colon were dissected into 3 separate layers (mucosa, lon-gitudinal, and circular muscles) and ileum samples were dissected into mucosa and muscle layers (n = 20). Complementary DNA was synthesized by reverse transcription from extracted RNA and expression was determined by quantitative or end point polymerase chain reaction. RESULTS: The 5-HT3 receptor subunits were found in all tissues throughout the colon and ileum. The A subunit was detected in all sam-ples and the C subunit was expressed at similar levels while the B subunit was expressed at lower levels and less frequently. The 5-HT3 receptor E subunit was mainly found in the mucosa layers. All splice variants of the 5-HT4 and 5-HT7 receptors were expressed throughout the colon although the 5-HT4 receptor d, g, and i variants were expressed less often. CONCLUSIONS: The major differences in 5-HT receptor distribution within the human colon are in relation to the mucosa and muscular tissue layers where the 5-HT3 receptor E subunit is predominantly found in the mucosal layer which may be of therapeutic relevance.