Kawashima E, Estoppey D, Virginio C, Fahmi D, Rees S, Surprenant A, North R A
Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development, Switzerland.
Recept Channels. 1998;5(2):53-60.
The 5' untranslated leader sequence from the encephalomyocarditis virus was used to engineer bicistronic or tricistronic expression vectors encoding two subunits (P2X2 and P2X3) of an ATP-gated cation channel. Human embryonic kidney (293) and chinese hamster ovary (CHO-K1) cells were transfected with the vector, and stable cell lines were generated by single cell subcloning. Selection was made in a 96-well format on the basis of a sustained increase in intracellular calcium (fluorescence of Fluo3-loaded cells) evoked by the ATP analog alpha beta methylene ATP. A high proportion of transformants expressed heteromeric receptors containing both P2X2 and P2X3 subunits, as evidenced by a nondesensitizing current in response to alpha beta methylene ATP. The method is fast and simple and could be generally useful for the stable expression of heteromultimeric channel proteins.
利用脑心肌炎病毒的5'非翻译前导序列构建了双顺反子或三顺反子表达载体，用于编码ATP门控阳离子通道的两个亚基（P2X2和P2X3）。将该载体转染到人胚肾（293）细胞和中国仓鼠卵巢（CHO-K1）细胞中，并通过单细胞亚克隆产生稳定细胞系。基于ATP类似物αβ亚甲基ATP诱发的细胞内钙持续增加（负载Fluo3的细胞的荧光），在96孔板中进行筛选。高比例的转化子表达同时含有P2X2和P2X3亚基的异源寡聚受体，这可通过对αβ亚甲基ATP产生的非脱敏电流得以证明。该方法快速且简单，可能普遍适用于异源多聚体通道蛋白的稳定表达。
