title: Characterization of the effects of four HTR3B polymorphisms on human 5-HT3AB receptor expression and signalling.
abstract: BACKGROUND
  5-Hydroxytryptamine 3 (5-HT3) receptors mediate the fast excitatory neurotransmission of serotonin. In this study, we have characterized the effects of four naturally occurring, nonsynonymous variants of the human 5-HT3B subunit on expression and signalling properties of heteromeric 5-HT3AB receptors.
  
  
  RESULTS
  5-HT3AB receptor signalling was studied in a fluorescence-based cell membrane potential assay and by electrophysiology. Expression levels of cotransfected epitope-tagged 5-HT3A and 5-HT3B subunits were determined using enzyme-linked immunosorbent assay and immunocytochemistry. In cells coexpressing 5-HT3A and 5-HT3B(I143T) subunits, cell surface expression levels of 5-HT3B in particular, and also 5-HT3A were markedly reduced compared with those of wild-type (WT) 5-HT3AB receptor-expressing cells. Electrophysiological recordings on cells coexpressing 5-HT3A and 5-HT3B(I143T) indicated cell surface expression of 5-HT3AB(I143T) receptors with macroscopic current kinetics similar to those of WT 5-HT3AB receptors but with 3-fold lower current densities. In the membrane potential assay, 5-HT3AB(I143T)-transfected cells exhibited signalling properties intermediate to those of WT 5-HT3AB and 5-HT3A receptors. Cotransfection of 5-HT3A, 5-HT3AB(I143T) and WT 5-HT3AB subunit cDNAs did not increase cell surface expression of the variant subunit nor did it restore WT 5-HT3AB receptor signalling completely in the membrane potential assay. In contrast to 5-HT3B(I143T), the 5-HT3B variants S156R, V183I and A223T did not give rise to significant changes in 5-HT3AB receptor expression or signalling properties.
  
  
  CONCLUSIONS
  5-HT3B(I143T)-containing 5-HT3AB receptors display significantly reduced cell surface expression and different signalling properties compared with WT 5-HT3AB receptors. In contrast, three other 5-HT3B variants, S156R, V183I and A223T, do not appear to alter 5-HT3AB receptor expression or signalling.
authors.affiliation: Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
authors.forename: Karen
authors.initials: K
authors.lastname: Krzywkowski
authors.forename: Paul A
authors.initials: PA
authors.lastname: Davies
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Irving
authors.forename: Hans
authors.initials: H
authors.lastname: Bräuner-Osborne
authors.forename: Anders A
authors.initials: AA
authors.lastname: Jensen
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004594:Electrophysiology; D004797:Enzyme-Linked Immunosorbent Assay; D006579:Heterozygote; D006801:Humans; D007150:Immunohistochemistry; D050505:Mutant Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011110:Polymorphism, Genetic / Q000187:drug effects; D021122:Protein Subunits / Q000235:genetics; D011985:Receptors, Serotonin / Q000235:genetics*; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
chemical_list: C509409:HTR3A protein, human; C509410:HTR3B protein, human; D050505:Mutant Proteins; D021122:Protein Subunits; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; D012701:Serotonin