Krzywkowski Karen, Davies Paul A, Irving Andrew J, Bräuner-Osborne Hans, Jensen Anders A
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
Pharmacogenet Genomics. 2008 Dec;18(12):1027-40. doi: 10.1097/FPC.0b013e328310f950.
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.
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.
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.
5-羟色胺3（5-HT3）受体介导血清素的快速兴奋性神经传递。在本研究中，我们已对人5-HT3B亚基的四种天然存在的非同义变体对异源5-HT3AB受体的表达和信号特性的影响进行了表征。
在基于荧光的细胞膜电位测定和电生理学中研究了5-HT3AB受体信号传导。使用酶联免疫吸附测定和免疫细胞化学测定共转染的表位标记的5-HT3A和5-HT3B亚基的表达水平。在共表达5-HT3A和5-HT3B（I143T）亚基的细胞中，与表达野生型（WT）5-HT3AB受体的细胞相比，5-HT3B的细胞表面表达水平，特别是5-HT3A的细胞表面表达水平明显降低。对共表达5-HT3A和5-HT3B（I143T）的细胞进行的电生理记录表明，5-HT3AB（I143T）受体的细胞表面表达具有与WT 5-HT3AB受体相似的宏观电流动力学，但电流密度低3倍。在膜电位测定中，转染5-HT3AB（I143T）的细胞表现出介于WT 5-HT3AB和5-HT3A受体之间的信号特性。共转染5-HT3A、5-HT3AB（I143T）和WT 5-HT3AB亚基cDNA既未增加变体亚基的细胞表面表达，也未在膜电位测定中完全恢复WT 5-HT3AB受体信号传导。与5-HT3B（I143T）相反，5-HT3B变体S156R、V183I和A223T未引起5-HT3AB受体表达或信号特性的显著变化。
与WT 5-HT3AB受体相比，含5-HT3B（I143T）的5-HT3AB受体表现出明显降低的细胞表面表达和不同的信号特性。相反，其他三种5-HT3B变体S156R、V183I和A223T似乎不会改变5-HT3AB受体的表达或信号传导。
