不同性质氨基酸置换对I型电压依赖型钠通道功能和抗癫痫药物反应的决定作用及其临床相关性研究

Voltage-gated sodium channel type I (Nav1.1), which encoded by SCN1A gene, plays a critical role in brain functional activity and served as a target of some anti-epileptic drugs (AEDs). SCN1A is now the most relevant epilepsy gene. So far, more than 1000 SCN1A mutations associated with epilepsy were identified. Missense mutations account for 48% among them. Previous studies suggest that missense mutations located on pore regions lead to severe phenotypes and seizures aggravation induced by AEDs, due to the loss of function of sodium channel; whereas those located on non-pore regions are associated with milder phenotypes. However, we have identified a missense mutation E1632A in S3 segment of Nav1.1 in patients with severe Dravet syndrome, who presented seizure aggravation by AEDs. The underlying mechanism warrants further studies. It is suspected whether different amino acid substitutions on the site of Nav1.1 may result severe or mild functional changes and are associated phenotype severity. In present study, we design amino acid substitutions of different properties nature at the site of 1632, including E1632A indentified in our patient, and 5 other amino acid substitutions (E1632V, E1632G, E1632K, E1632Q and E1632D) based on the principle of single-base mutation. We plan to study the functional alterations of these mutants by using expressions of heterogonous cells in vitro, including detection of electrophysiological properties of sodium channel by using the whole cell patch clamp technique, and the expression level of SCN1A gene and Nav1.1 protein expressed in cell membrane. Subsequently, different types of AEDs with different dosage will be administrated to each mutant to observe their electrophysiological properties changes of sodium channel. Comprehensive results of in vitro experiments will be analyzed to explore the relationship among genotypes, phenotypes, functional types as well as AEDs responses. This study will help understand the mechanism of refractory epilepsy caused by SCN1A missense mutation in non-pore regions and disclose the effects of sub-molecular structure changes on the sodium channel function and AEDs reaction.

I 型电压依赖型钠通道（Nav1.1）由SCN1A基因编码，临床上与癫痫关系密切，同时是多种抗癫痫药物（AEDs）作用的靶点。现已发现一千多个癫痫有关的SCN1A突变，错义突变占48%。既往认为Nav1.1孔区错义突变可以引起通道功能丧失而导致严重表型及AEDs加重发作；非孔区错义突变则与较轻的表型有关。我们此前在严重癫痫患者中筛查到Nav1.1非孔区的错义突变E1632A，AEDs可加重其发作，但其潜在机制不清。因此，我们在该位点设计不同性质氨基酸置换，通过异源细胞体外表达，研究不同突变体的电生理特性变化，以及对SCN1A基因表达和Nav1.1胞膜上表达的影响。随后每个突变体分别给予不同类型和浓度AEDs处理，观察其通道电生理改变。综合体外研究结果，分析其与临床的可能关系。其结果将对揭钠通道非孔区错义突变导致顽固性癫痫的机制，亚分子结构改变对钠通道功能和AEDs疗效的的影响有重要意义。

I 型电压依赖型钠通道（Nav1.1）由SCN1A基因编码，临床上与癫痫关系密切，同时 是多种抗癫痫药物（AEDs）作用的靶点。现已发现一千多个癫痫有关的SCN1A突变，错义突变占48%。既往认为Nav1.1孔区错义突变可以引起通道功能丧失而导致严重表型及AEDs加重发作；非孔区错义突变则与较轻的表型有关。我们此前在严重癫痫患者中筛查到Nav1.1非孔区的错义突变E1632A，AEDs可加重其发作，但其潜在机制不清。因此，我们在该位点设计不同性质氨基酸置换，通过异源细胞体外表达，研究不同突变体的电生理特性变化，以及对SCN1A基因表达和Nav1.1胞膜上表达的影响。结果发现Nav1.1 通道蛋白第1623位氨基酸可能以极性变化对通道电生理动力学性质影响最大，表现为E1623A、E1623V突变体电流密度降低，激活速度减慢，更容易失活，失活后恢复速度减慢。Nav1.1 通道功能降低，表现为“pLOF”。第1623位氨基酸所带电荷改变、结构变化及分子量大小也对Nav1.1 通道功能有一定影响。

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