Pure haploinsufficiency for Dravet syndrome NaV1.1 (SCN1A) sodium channel truncating mutations
Pure haploinsufficiency for Dravet syndrome NaV1.1 (SCN1A) sodium channel truncating mutations
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DOI：
10.1111/j.1528-1167.2011.03346.x
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发表时间：
2012-01-01
期刊：
影响因子：
5.6
通讯作者：
Mantegazza, Massimo
中科院分区：
文献类型：
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作者：
Bechi, Giulia;Scalmani, Paolo;Mantegazza, Massimo
目的：Dravet综合征(DS)是一种毁灭性的癫痫性脑病，主要由编码电压门控Na+通道Nav1.1a亚单位的SCN1A基因突变引起。大约50%的SCN1ADS突变截断了Nav1.1，可能导致其功能的完全丧失。然而，目前还没有研究表明，如果Nav1.1截断突变体损害了野生型通道的表达或功能，那么它们是否为显性负性，就像对其他蛋白质(如CAV通道)的截断突变体所显示的那样。方法：在人Na(V)1.1(HNA(V)1.1)的cDNA中构建R222*或R1234*，并研究其对共表达野生型HNA(V)1.1、HNA(V)1.2或HNA(V)1.3共表达TSA-201细胞的影响，以及对稳定表达该通道的人胚胎肾(HEK)细胞系HNA(V)1.6的影响。我们还研究了从Na(V)1.1基因敲除(KO)小鼠分离的海马神经元，这是一种表达截短的Na(V)1.1通道的动物模型。关键发现：我们发现与HNA(V)1.1、HNA(V)1.2或HNA(V)1.3共表达的截短突变体的电流幅度没有改变，但与HNA(V)1.6共表达的电流幅度降低了30%。然而，我们发现，共表达功能性全长HNA(V)1.1也会导致类似的减少。因此，这一效应不应参与DS的发病机制。HNA(V)1.1、HNA(V)1.3和HNA(V)1.6的某些门控特性被修改，但从Na(V)1.1 KO小鼠分离的海马神经元的记录没有显示出任何显著的修改。因此，Na(V)1.1截短突变体不是显性负的，与单倍体不足是DS的原因相一致。意义：我们更好地阐明了DS的发病机制，指出了致病截短CaV2.1突变体与HNA(V)1.1突变体的重要区别，并表明在生理条件下，共表达HNA(V)1.1可以降低HNA(V)1.6的表达。此外，我们的数据可能会为治疗方法的发展提供有用的信息。
Purpose: Dravet syndrome (DS), a devastating epileptic encephalopathy, is mostly caused by mutations of the SCN1A gene, coding for the voltage-gated Na+ channel NaV1.1 a subunit. About 50% of SCN1A DS mutations truncate NaV1.1, possibly causing complete loss of its function. However, it has not been investigated yet if NaV1.1 truncated mutants are dominant negative, if they impair expression or function of wild-type channels, as it has been shown for truncated mutants of other proteins (e.g., CaV channels). We studied the effect of two DS truncated NaV1.1 mutants, R222* and R1234*, on coexpressed wild-type Na+ channels.Methods: We engineered R222* or R1234* in the human cDNA of Na(V)1.1 (hNa(V)1.1) and studied their effect on coexpressed wild- type hNa(V)1.1, hNa(V)1.2 or hNa(V)1.3 cotransfecting tsA- 201 cells, and on hNa(V)1.6 transfecting an human embryonic kidney (HEK) cell line stably expressing this channel. We also studied hippocampal neurons dissociated from Na(V)1.1 knockout (KO) mice, an animal model ofDSexpressing a truncatedNa(V)1.1 channel.Key Findings: We found no modifications of current amplitude coexpressing the truncated mutants with hNa(V)1.1, hNa(V)1.2, or hNa(V)1.3, but a 30% reduction coexpressing them with hNa(V)1.6. However, we showed that also coexpression of functional full- length hNa(V)1.1 caused a similar reduction. Therefore, this effect should not be involved in the pathomechanism of DS. Some gating properties of hNa(V)1.1, hNa(V)1.3, and hNa(V)1.6 were modified, but recordings of hippocampal neurons dissociated from Na(V)1.1 KO mice did not show any significant modifications of these properties. Therefore, Na(V)1.1 truncated mutants are not dominant negative, consistent with haploinsufficiency as the cause of DS.Significance: We have better clarified the pathomechanism of DS, pointed out an important difference between pathogenic truncated CaV2.1 mutants and hNa(V)1.1 ones, and shown that hNa(V)1.6 expression can be reduced in physiologic conditions by coexpression of hNa(V)1.1. Moreover, our data may provide useful information for the development of therapeutic approaches.