Tan Jianguo, Liu Zhiqi, Nomura Yoshiko, Goldin Alan L, Dong Ke
Department of Entomology and Neuroscience Program, Michigan State University, East Lansing, Michigan 48824, USA.
J Neurosci. 2002 Jul 1;22(13):5300-9. doi: 10.1523/JNEUROSCI.22-13-05300.2002.
Alternative splicing is a major mechanism by which potassium and calcium channels increase functional diversity in animals. Extensive alternative splicing of the para sodium channel gene and developmental regulation of alternative splicing have been reported in Drosophila species. Alternative splicing has also been observed for several mammalian voltage-gated sodium channel genes. However, the functional significance of alternative splicing of sodium channels has not been demonstrated. In this study, we identified three mutually exclusive alternative exons encoding part of segments 3 and 4 of domain III in the German cockroach sodium channel gene, para(CSMA). The splice site is conserved in the mouse, fish, and human Na(v)1.6 sodium channel genes, suggesting an ancient origin. One of the alternative exons possesses a stop codon, which would generate a truncated protein with only the first two domains. The splicing variant containing the stop codon is detected only in the PNS, whereas the other two full-size variants were detected in both the PNS and CNS. When expressed in Xenopus oocytes, the two splicing variants produced robust sodium currents, but with different gating properties, whereas the splicing variant with the stop codon did not produce any detectable sodium current. Furthermore, these two functional splicing variants exhibited a striking difference in sensitivity to a pyrethroid insecticide, deltamethrin. Exon swapping partially reversed the channel sensitivity to deltamethrin. Our results therefore provide the first evidence that alternative splicing of a sodium channel gene produces pharmacologically distinct channels.
可变剪接是钾离子和钙离子通道增加动物功能多样性的一种主要机制。在果蝇物种中，已报道了para钠通道基因广泛的可变剪接以及可变剪接的发育调控。在几种哺乳动物电压门控钠通道基因中也观察到了可变剪接。然而，钠通道可变剪接的功能意义尚未得到证实。在本研究中，我们在德国小蠊钠通道基因para(CSMA)中鉴定出三个相互排斥的可变外显子，它们编码结构域III的第3和第4段的部分区域。该剪接位点在小鼠、鱼类和人类Na(v)1.6钠通道基因中保守，表明其起源古老。其中一个可变外显子含有一个终止密码子，这将产生一个仅具有前两个结构域的截短蛋白。含有终止密码子的剪接变体仅在周围神经系统中检测到，而其他两个全长变体在周围神经系统和中枢神经系统中均有检测到。当在非洲爪蟾卵母细胞中表达时，这两个剪接变体产生了强大的钠电流，但具有不同的门控特性，而含有终止密码子的剪接变体未产生任何可检测到的钠电流。此外，这两个功能性剪接变体对拟除虫菊酯类杀虫剂溴氰菊酯的敏感性表现出显著差异。外显子交换部分逆转了通道对溴氰菊酯的敏感性。因此，我们的结果首次证明了钠通道基因的可变剪接产生了药理学上不同的通道。
