Baculovirus Expression Provides Direct Evidence for Heteromeric Assembly of P2X2 and P2X3 Receptors
Baculovirus Expression Provides Direct Evidence for Heteromeric Assembly of P2X2 and P2X3 Receptors
复制标题
DOI：
10.1523/jneurosci.17-17-06529.1997
复制
发表时间：
1997-09
期刊：
影响因子：
--
通讯作者：
Kathryn M. Radford;C. Virginio;A. Surprenant;R. North;E. Kawashima
中科院分区：
文献类型：
--
作者：
Kathryn M. Radford;C. Virginio;A. Surprenant;R. North;E. Kawashima
P2X2和P2X3是P2X受体的亚基，通过结合细胞外ATP打开阳离子通道。编码P2X2和P2X3受体亚基的cdna分别具有两个c端表位标签中的一个，被克隆到杆状病毒中。病毒感染的昆虫细胞（Spodoptera frugiperda）通过[35S]ATP的特异性结合和ATP或αβ亚甲基ATP引起的全细胞膜电流记录，通过Western blotting检测到相应蛋白的表达中至高水平。在同时被编码P2X2和P2X3受体的两种病毒感染的细胞中，这两种蛋白可以与任何一种表位标签特异性的抗体交叉免疫沉淀。这些细胞的全细胞记录显示，ATP和αβ -亚甲基ATP引起的电流具有激动剂敏感性和脱敏性，与单独表达P2X2或P2X3受体时观察到的结果截然不同。该结果提供了一种表达大量P2X受体蛋白的方法，并提供了直接证据，证明P2X2和P2X3亚基组装形成异质通道，与形成的同质通道具有不同的性质。
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 αβmethylene-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 αβmethylene-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.