Radford K M, Virginio C, Surprenant A, North R A, Kawashima E
Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development, 1228 Geneva, Switzerland.
J Neurosci. 1997 Sep 1;17(17):6529-33. doi: 10.1523/JNEUROSCI.17-17-06529.1997.
P2X2 and P2X3 are subunits of P2X receptors, cation channels opened by binding extracellular ATP. cDNAs encoding P2X2 and P2X3 receptor subunits, each with one of two C-terminal epitope tags, were cloned into baculovirus. Virally infected insect cells (Spodoptera frugiperda) expressed moderate to high levels of the corresponding proteins, as detected by Western blotting, by the specific binding of [35S]ATP and by whole-cell recordings of membrane current evoked by ATP or alphabetamethylene-ATP. In cells infected at the same time with two viruses encoding P2X2 and P2X3 receptors, the two proteins could be cross-immunoprecipitated with antibodies specific for either of the epitope tags. Whole-cell recordings from these cells showed that ATP and alphabetamethylene-ATP evoked currents with agonist sensitivity and desensitization quite distinct from those observed when P2X2 or P2X3 receptors were expressed alone. The results offer a method to express large amounts of P2X receptor protein, and they provide direct evidence that P2X2 and P2X3 subunits assemble to form heteromeric channels having distinct properties from those formed as homomers.
P2X2和P2X3是P2X受体的亚基，P2X受体是由细胞外ATP结合而开启的阳离子通道。编码P2X2和P2X3受体亚基的cDNA（每个都带有两个C末端表位标签中的一个）被克隆到杆状病毒中。通过蛋白质印迹法、[35S]ATP的特异性结合以及ATP或α,β-亚甲基-ATP诱发的膜电流的全细胞记录检测到，被病毒感染的昆虫细胞（草地贪夜蛾）表达了中等至高水平的相应蛋白质。在同时被两种编码P2X2和P2X3受体的病毒感染的细胞中，这两种蛋白质可以用针对任一表位标签的特异性抗体进行交叉免疫沉淀。来自这些细胞的全细胞记录表明，ATP和α,β-亚甲基-ATP诱发的电流，其激动剂敏感性和脱敏作用与单独表达P2X2或P2X3受体时观察到的情况截然不同。这些结果提供了一种表达大量P2X受体蛋白的方法，并且它们提供了直接证据，证明P2X2和P2X3亚基组装形成了具有与同聚体形成的通道不同特性的异聚体通道。
