Abstract
Within the family of serotonin receptors, the 5-hydroxytryptamine-3 (5-HT3) receptor is the only ligand-gated ion channel. It is composed of five subunits, of which the 5-HT3A and 5-HT3B subunits are best characterized. Several studies, however, have reported on the functional diversity of native 5-HT3 receptors, which cannot solely be explained on the basis of the 5-HT3A and 5-HT3B subunits. After our discovery of further putative 5-HT3 serotonin receptor-encoding genes, HTR3C, HTR3D, and HTR3E, we investigated whether these novel candidates and the isoform 5-HT3Ea are able to form functional 5-HT3 receptor complexes. Using immunofluorescence and immunoprecipitation studies of heterologously expressed proteins, we found that each of the respective candidates coassembles with 5-HT3A. To investigate whether the novel subunits modulate 5-HT3 receptor function, we performed radioligand-binding assays and calcium-influx studies in human embryonic kidney 293 cells. Our experiments revealed that the 5-HT3C,5-HT3D, 5-HT3E, and 5-HT3Ea subunits alone cannot form functional receptors. Coexpression with 5-HT3A, 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-HT3A and heteromeric complexes. However, 5-HT showed increased efficacy with respect to 5-HT3A/D and 5-HT3A/E receptors, which is consistent with the increased surface expression compared with 5-HT3A receptors. In contrast, 5-HT3A/C and 5-HT3A/Ea receptors exhibited decreased 5-HT efficacy. These data show for the first time that the novel 5-HT3 subunits are able to form heteromeric 5-HT3 receptors, which exhibit quantitatively different functional properties compared with homomeric 5-HT3A receptors.
Footnotes
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This study was supported by the German Federal Ministry for Education and Research in the National Genome Research Network NGFN-2 EP (B.N.) and the Deutsche Forschungsgemeinschaft (M.B. and M.G.). Parts of this work were presented previously at the SfN Neuroscience Meeting 2006 in Atlanta, GA, as a poster presentation (number 625.5).
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ABBREVIATIONS: 5-HT, 5-hydroxytryptamine, serotonin; LGIC, ligand-gated ion channel; HEK, human embryonic kidney; mCPBG, meta-chlorophenylbiguanide; GI, gastrointestinal; HA, hemagglutinin; PBS, phosphate-buffered saline; ANOVA, analysis of variance; FITC, fluorescein isothiocyanate; RLU, relative light unit; PVDF, polyvinylidene difluoride; ER, endoplasmic reticulum; HRP, horseradish peroxidase; MDL72222, 3-tropanyl-3,5-dichlorobenzoate; Y-25130, azasetron; [3H]GR65630, [3H]3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received October 24, 2006.
- Accepted March 28, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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