No abstract

Within the family of serotonin receptors, the 5-hydroxytryptamine-3 (5-HT 3 ) receptor is the only ligand-gated ion channel. It is composed of five subunits, of which the 5-HT 3A and 5-HT 3B subunits are best characterized. Several studies, however, have reported on the functional diversity of native 5-HT 3 receptors, which cannot solely be explained on the basis of the 5-HT 3A and 5-HT 3B subunits. After our discovery of further putative 5-HT 3 serotonin receptor-encoding genes, HTR3C, HTR3D, and HTR3E, we investigated whether these novel candidates and the isoform 5-HT 3Ea are able to form functional 5-HT 3 receptor complexes. Using immunofluorescence and immunoprecipitation studies of heterologously expressed proteins, we found that each of the respective candidates coassembles with 5-HT 3A . To investigate whether the novel subunits modulate 5-HT 3 receptor function, we performed radioligandbinding assays and calcium-influx studies in human embryonic kidney 293 cells. Our experiments revealed that the 5-HT 3C , 5-HT 3D , 5-HT 3E , and 5-HT 3Ea subunits alone cannot form functional receptors. Coexpression with 5-HT 3A , however, results in the formation of functional heteromeric complexes with different serotonin efficacies. Potencies of two agonists and antagonists were nearly identical with respect to homomeric 5-HT 3A and heteromeric complexes. However, 5-HT showed increased efficacy with respect to 5-HT 3A/D and 5-HT 3A/E receptors, which is consistent with the increased surface expression compared with 5-HT 3A receptors. In contrast, 5-HT 3A/C and 5-HT 3A/Ea receptors exhibited decreased 5-HT efficacy. These data show for the first time that the novel 5-HT 3 subunits are able to form heteromeric 5-HT 3 receptors, which exhibit quantitatively different functional properties compared with homomeric 5-HT 3A receptors.The 5-HT 3 receptor is the only ligand-gated ion channel (LGIC) within the family of serotonin (5-hydroxytryptamine, 5-HT) receptors (Hoyer et al., 2002). Based on structural and functional homologies, the nicotinic acetylcholine receptor and the 5-HT 3 receptor are most closely related; both are cation channels. The 5-HT 3 receptor is formed by a pentameric complex and is permeable to Na ϩ , K ϩ , and Ca 2ϩ . Binding of serotonin to the 5-HT 3 receptor leads to a fast excitatory response of the neuron. After cloning of the human HTR3A gene (Belelli et al., 1995;Miyake et al., 1995), findings concerning variable receptor compositions and properties led to the hypothesis that further 5-HT 3 receptor subunits and isoforms should exist (Hussy et al., 1994;Jackson and Yakel, 1995;Fletcher and Barnes, 1998). This hypothesis was confirmed by the cloning of the human HTR3B gene (Davies et al., 1999) and of two different human splice variants of the HTR3A gene (Brü ss et al., 2000). To date, HTR3A and HTR3B (Belelli et al., 1995;Miyake et al., 1995;Davies et al., 1999) are well characterized. 5-HT 3A subunits are able to form functional homooligomeric receptors after expression in Xenopus laevi...

Diarrhea predominant irritable bowel syndrome (IBS-D) is a complex disorder related to dysfunctions in the serotonergic system. As cis-regulatory variants can play a role in the etiology of complex conditions, we investigated the untranslated regions (UTRs) of the serotonin receptor type 3 subunit genes HTR3A and HTR3E. Mutation analysis was carried out in a pilot sample of 200 IBS patients and 100 healthy controls from the UK. The novel HTR3E 3'-UTR variant c.*76G>A (rs62625044) was associated with female IBS-D (P = 0.033, OR = 8.53). This association was confirmed in a replication study, including 119 IBS-D patients and 195 controls from Germany (P = 0.0046, OR = 4.92). Pooled analysis resulted in a highly significant association of c.*76G>A with female IBS-D (P = 0.0002, OR = 5.39). In a reporter assay, c.*76G>A affected binding of miR-510 to the HTR3E 3'-UTR and caused elevated luciferase expression. HTR3E and miR-510 co-localize in enterocytes of the gut epithelium as shown by in situ hybridization and RT-PCR. This is the first example indicating micro RNA-related expression regulation of a serotonin receptor gene with a cis-regulatory variant affecting this regulation and appearing to be associated with female IBS-D.

Our findings may encourage the use of ginger extracts to alleviate nausea in cancer patients receiving chemotherapy and to treat functional GI disorders.

Oxymetazoline and xylometazoline are both used as nasal mucosa decongesting α-adrenoceptor agonists during a common cold. However, it is largely unknown which of the six α-adrenoceptor subtypes are actually present in human nasal mucosa, which are activated by the two alpha-adrenoceptor agonists and to what extent. Therefore, mRNA expression in human nasal mucosa of the six α-adrenoceptor subtypes was studied. Furthermore, the affinity and potency of the imidazolines oxymetazoline and xylometazoline at these α-adrenoceptor subtypes were examined in transfected HEK293 cells. The rank order of mRNA levels of α-adrenoceptor subtypes in human nasal mucosa was: α(2A) > α(1A) ≥ α(2B) > α(1D) ≥ α(2C) >> α(1B) . Oxymetazoline and xylometazoline exhibited in radioligand competition studies higher affinities than the catecholamines adrenaline and noradrenaline at most α-adrenoceptor subtypes. Compared to xylometazoline, oxymetazoline exhibited a significantly higher affinity at α(1A) - but a lower affinity at α(2B) -adrenoceptors. In functional studies in which adrenoceptor-mediated Ca(2+) signals were measured, both, oxymetazoline and xylometazoline behaved at α(2B) -adrenoceptors as full agonists but oxymetazoline was significantly more potent than xylometazoline. Furthermore, oxymetazoline was also a partial agonist at α(1A) -adrenoceptors; however, its potency was relatively low and it was much lower than its affinity. The higher potency at α(2B) -adrenoceptors, i.e. at receptors highly expressed at the mRNA level in human nasal mucosa, could eventually explain why in nasal decongestants oxymetazoline can be used in lower concentrations than xylometazoline.

The demonstrated 5-HT3 inhibitory effects may be relevant for boldine's and menthol's alleviating properties on FGID and may encourage clinical studies with the compounds or the plant extracts for CINV and IBS treatment. The found receptor-discriminative properties make boldine and (-)-menthol to potentially useful tools for analyzing structural differences between these receptor subtypes.

These results may help to explain earlier reported association findings of the frequent p.Y129S and p.V183I variants with psychiatric diseases. Replication studies with larger sample pools, especially regarding the rare p.S156R variant would be useful, to obtain an idea about the predisposing role of these single nucleotide polymorphisms as susceptibility variants.

Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A–E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT3 receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT3 receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca2+ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT3A receptors but leads to differential increases of 5-HT-induced maximum response (Emax) on cells expressing different subunits. Increases of Emax were due to analogously enhanced Bmax values for binding of the 5-HT3 receptor antagonist [3H]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT3A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT3 receptor composition in vivo.