【作者】 刘超；

【导师】 廖卫平；

【作者基本信息】 广州医学院 ， 内科学， 2011， 博士

【摘要】 研究背景癫痫是脑部神经元高度同步化异常放电所致,以发作性、短暂性、重复性及刻板性为特征的一组中枢神经系统功能失常的综合征。神经元细胞膜兴奋性增高,易于形成痫性放电,是癫痫发生的病理生理基础。研究发现了越来越多的单基因突变导致癫痫,这些基因大多数编码离子通道或受体,包括电压门控钠、钾、钙、氯离子通道和乙酰胆碱受体及γ-氨基丁酸受体(GABA)。电压门控钠通道主要负责控制细胞兴奋性,其结构和功能异常可引起细胞膜兴奋性改变,在癫痫发病机制中具有重要作用。哺乳动物的电压门控钠通道由α亚基和β亚基组成。α亚基由同一家族的9个基因编码(SCN1A-SCN11A),SCN1A基因编码的蛋白命名为Nav1.1,Nav1.1主要表达在抑制性中间神经元。α亚基是钠通道的功能性单位,由四个高度同源性的结构域(Ⅰ～Ⅳ)通过胞内连接环相连而成,每个结构域含有6个跨膜片段(S1～S6)。4个辅助性β亚基(β1-β4)由基因SCN1B-SCN4B基因编码,在成人中枢神经系统α亚基一般连有β1和β2亚基。SCN1A基因突变主要导致三种癫痫综合症:全面性癫痫伴热性惊厥附加症(generalized epilepsy with febrile seizures plus, GEFS+),婴儿重症肌阵挛癫痫(severe myoclonic epilepsy of infancy, SMEI)和部分性癫痫伴热性惊厥附加症(partial epilepsy with febrile seizures plus, PEFS+)。SCN1B基因的突变主要导致GEFS+。我们在对SMEI患者相关基因筛查中,发现了一个新的SCN1A基因错义突变:c.4868A>C,导致Nav1.1第1623位氨基酸由谷氨酸变为丙氨酸(E1623A),位于Nav1.1第4个结构域S3片断上,而且高度保守。目前,在SMEI患者Nav1.1 S3跨膜片共发现10个突变,但这些突变对钠通道功能的影响还未见报道,Nav1.1E1623A导致癫痫的发病机制值得进一步研究。研究目的通过在HEK 293T细胞异质表达Nav1.1 E1623A和β1和β2亚基,采用全细胞膜片钳记录钠通道的电生理动力学参数,将其与Nav1.1野生型进行比较,探讨SCN1A基因c.4868A>C突变导致癫痫的发病机制。研究方法1. pCMV-SCN1A质粒酶切、测序及Nav1.1表达的鉴定采用内切酶消化pCMV-SCN1A质粒初步鉴定,酶切正确的质粒测序确认SCN1A基因编码区。HEK 293T细胞转染pCMV-SCN1A质粒48h后提取总蛋白,采用蛋白印迹检测Nav1.1的表达。2. pDsred-IRES-SCN1B质粒构建和鉴定采用引入酶切位点的PCR方法从pCMV-Dsred质粒扩增红色荧光蛋白Dsred DNA片段。将Dsred PCR产物克隆到TA载体,测序确认。在pCD8-IRES-SCN1B质粒的基础上内切酶消化去除pCD8标记,回收IRES-SCN1B载体;pDsred-TA质粒内切酶消化回收Dsred DNA片段,采用DNA连接酶连接Dsred DNA片段与IRES-SCN1B载体,即得pDsred-IRES-SCN1B质粒,测序确认SCN1B基因编码区。3. pGFP-IRES-SCN2B质粒酶切、测序采用内切酶消化pGFP-IRES-SCN2B质粒初步鉴定,酶切正确的质粒测序确认SCN2B基因编码区。4. pCMV-SCN1A-E1623A突变体质粒的构建定点诱变试剂盒对pCMV-SCN1A质粒进行定点诱变构建pCMV-SCN1A- E123A质粒,测序确认。5.膜片钳检测HEK 293T细胞共转染pDsred-IRES-SCN1B质粒、pGFP-IRES-SCN2B质粒和pCMV-SCN1A质粒或pCMV-SCN1A-E1623A质粒48h后,在荧光显微镜下可观察到红色荧光和绿色荧光。选择同时表达了红色荧光和绿色荧光的HEK293T细胞进行电生理功能研究。在全细胞电压钳模式下给予标准刺激方案,根据所得钠电流数据建立钠通道的电流密度曲线、电压依赖激活曲线、稳态失活曲线和失活后恢复曲线,计算钠通道的电流密度、半数激活电压、半数失活电压和失活后恢复时间常数等进行比较。6.统计学分析计量资料以均数±标准差( X±S)表示,采用SPSS l6.0软件包进行统计分析,组间比较采用两样本t检验。P<0.05为差异具有统计学意义。研究结果1. pCMV-SCN1A质粒酶切、测序及Nav1.1表达的检测pCMV-SCN1A质粒测序SCN1A基因编码区未发现突变。Western blot检测结果显示:HEK 293T细胞转染pCMV-SCN1A质粒48h后在蛋白水平得到明显表达。2. pDsred-IRES-SCN1B质粒构建和鉴定成功将红色荧光蛋白Dsred DNA片段连接到IRES-SCN1B载体。pDsred-IRES-SCN1B质粒测序SCN1B基因编码区未发现突变。3. pGFP-IRES-SCN2B质粒酶切、测序测序确认pGFP-IRES-SCN2B质粒SCN2B基因编码区未发现突变。4. pCMV-SCN1A-E1623A突变体质粒的构建测序证实:SCN1A基因第1623位谷氨酸密码子(GAG)突变为丙氨酸密码子(GCG),而该质粒上SCN1A基因其它区段均未出现意外突变。5.膜片钳检测Nav1.1野生型和E1623A突变体在去极化电压为-10mv时,钠通道的电流最大。Nav1.1野生型峰电流密度约为-232.1±19.1pA/pF,激活曲线半数激活电位(V1/2)为-24.8±0.4mv(n=10),斜率因子K为5.2±0.3;稳态失活曲线半数失活电位(V1/2)为-53.0±0.5mv(n=10),斜率因子K为-8.6±0.4;失活后恢复时间常数τfast和τslow分别为1.4±0.1ms(n=9)和39.9±6.0ms。与Nav1.1野生型相比,E1623A突变体峰电流密度约为-100.7±16.2pA/pF,明显降低(p<0.05)。激活曲线半数激活电位(V1/2)为-24.6±0.5mv(n=7),斜率因子K为8.4±0.4(p<0.05),激活速度减慢;稳态失活曲线向超极化方向移动,半数失活电位(V1/2)为-60.9±0.5mv(n=7)(p<0.05),斜率因子K为-11.5±0.5(p<0.05),失活加快;失活后恢复时间常数τfast和τslow分别为2.1±0.2m(sn=6)(p<0.05)和42.5±6.9ms,失活后恢复时间延长。结论1. Nav1.1 E1623A突变体的电流密度降低,激活速度减慢,失活加快,失活后恢复时间延长,Nav1.1 E1623A突变体功能减弱,表现为“partial loss of function”。2. Nav1.1 E1623A突变体可能导致抑制性中间神经元Nav1.1功能减弱,对兴奋性神经元抑制作用降低,从而引起兴奋性神经元兴奋性增高,这可能是该患者癫痫发病的主要机制,可为该患者选择抗癫痫药物提供参考。更多还原

【Abstract】 BackgroundEpilepsy is a group of central nervous system dysfunction syndrome with the alteration of neuronal excitability, a high degree of synchronization of abnormal brain neurons discharging, and characterized as episodic, transient, repetitive and stereotyped. Epileptic discharge is easily formed in the case of increasing membrane excitability of neurons, which is the basis of the pathophysiology of epilepsy. Research has uncovered a growing number of single-gene mutations that cause epilepsy. The majority of these genes encode ion channels or receptors, including voltage-gated sodium, potassium, calcium, chloride channels and acetylcholine receptors andγ-aminobutyric acid (GABA) receptor. The voltage-gated sodium channels are in part responsible for controlling cell electrical excitability, and its abnormal structure and function can cause changes in membrane excitability, which play an important role in the pathogenesis of epilepsy.The mammalian voltage-gated sodium channel consists ofαsubunit and twoβsubunits. Theαsubunits are encoded by nine genes of the same family (SCN1A-SCN11A), SCN1A encode one isoform, termed Nav1.1. Theαsubunit comprises four homologous domains termed I–IV. Within each domain, there are six transmembrane segments called S1–S6. The Nav1.1 is predominantly expressed in inhibitory interneurons. Four accessoryβ1,β2,β3, orβ4 subunits are encoded by SCN1B-SCN4B respectively. In the adult CNS, theαsubunits are associated withβ1 andβ2. SCN1A mutations mainly can cause the three epilepsy syndrome, generalized epilepsy with febrile seizures plus (GEFS+), severe myoclonic epilepsy of infancy (SMEI), and partial epilepsy with febrile seizures plus (PEFS+). Mutations in SCN1B lead to GEFS+. We found a novel missense mutation c.4868A>C in SCN1A genetic screening of patients with SMEI. The mutation lead to the 1623 amino acid glutamate into alanine (E1623A), it was located at S3 segments of the fourth domain where highly conserved. Up to now, there are 10 mutations located in S3 of Nav1.1 in patients with SMEI, but the effect of these mutations on the function of Nav1.1 has not been reported. The mechanism of Nav1.1 E1623A leading to epilepsy is worthy of further study.PurposeTo explore the pathogenesis of epilepsy caused by the mutation of SCN1A c.4868 A>C. by comparing the electrophysiological changes using whole cell patch clamp in HEK 293T cells with expression of Nav1.1 E1623A andβ1 andβ2 subunits.Methods1. Sequencing the pCMV-SCN1A plasmid and identificating the expression of Nav1.1pCMV-SCN1A plasmid was identified first by endonuclease, and then the SCN1A coding region was confirmed by sequencing. Total protein of HEK 293T cells transfected with pCMV-SCN1A plasmid was extracted after 48h, and the expression of Nav1.1 was detected by using western blot.2. Constructing the pDsred-IRES-SCN1B plasmidRed fluorescent DNA fragment was amplified from pCMV-Dsred plasmid using PCR methods with disigned restriction enzyme cutting site, and it was cloned to TA vector. pDsred-TA plasmid which was confirmed by sequencing was digested with endonuclease, and Dsred DNA fragment was gotten. pCD8 marks was removed from pCD8-IRES-SCN1B plasmid by endonuclease and IRES-SCN1B vector was gotten. pDsred-IRES-SCN1B plasmid was constructed by ligating Dsred DNA fragment with IRES-SCN1B vector with DNA ligase. SCN1B coding region in pDsred-IRES- SCN1B plasmid was confirmed by sequencing. 3. Sequencing the pGFP-IRES-SCN2B plasmidpGFP-IRES-SCN2B plasmid was identified first by endonuclease, and then the SCN2B coding region was confirmed by sequencing.4. Constructing the pCMV-SCN1A-E1623A plasmidpCMV-SCN1A-E123A plasmid was constructed with site-directed mutagenesis kit and confirmed by sequencing.5. Patch clamp detectionpCMV-SCN1A plasmid or pCMV-SCN1A-E1623A plasmid, pDsred-IRES- SCN1B plasmid and pGFP-IRES-SCN2B plasmid were co-transfected in HEK 293T cells, the red and green fluorescence were observed under fluorescent microscope after 48h. Only cell with the red and green fluorescence can be used for next patch clamp detection. During patch clamp detection, the standard stimulus was given in the whole-cell voltage clamp mode. According to current data, the current density curves of sodium current, voltage dependent activation curve, steady-state inactivation curve and recovery curve after inactivation were established, and the current density of sodium current, half activation voltage, half inactivation voltage and resurrection time constant were calculated and compared.6. Statistical analysisData were shown as mean±standard deviation ( X±S). SPSS l6.0 software package was used for the statistical methods. Statistical comparisons were done with the two-sample t-test. P <0.05 was statistically significant difference.Results1. Sequencing the pCMV-SCN1A plasmid and identificating the expression of Nav1.1No mutation was found in coding sequence of SCN1A in pCMV-SCN1A plasmid. Western blot results showed that: the level of Nav1.1 has been markedly expressed in HEK 293T cells transfected with pCMV-SCN1A plasmids 48h later.2. Constructing the pDsred-IRES-SCN1B plasmidDsred PCR products were successfully ligated to the IRES-SCN1B vector. pDsred-IRES-SCN1B plasmid was sequenced and no mutation was found in its coding region.3. Sequencing the pGFP-IRES-SCN2B plasmidNo mutation was found in coding sequence of SCN2B in pGFP-IRES-SCN2B plasmid.4. Constructing the pCMV-SCN1A-E1623A plasmidThe mutation of 1623 glutamic acid codon (GAG) to alanine codon (GCG) in SCN1A was confirmed by sequencing, while other mutations in SCN1A in the plasmid were not found.5. Patch clamp detectionAt -10mv, the level of sodium channel current was maximal in wild type Nav1.1 and E1623A mutant. Peak current density of wild type Nav1.1 is about -232.1±19.1pA/pF (n=10), half activation potential (V1/2) of activation curve was -24.8±0.4mv (n=10), slope factor K was 5.2±0.3; half inactivation potential (V1/2) of steady-state inactivation curve was -53.0±0.5mv (n=10), slope factor K was -8.6±0.4; Recovery time constantτfast andτslow were 1.4±0.1ms (n=9) and 39.9±6.0ms .Compared with that of wild type Nav1.1, peak current density of E1623A mutant was about -100.7±16.2pA/pF (n=7) (p <0.05); half activation potential (V1/2) of activation curve was -24.6±0.5mv (n=7), slope factor K is 8.4±0.4 (p<0.05); steady-state inactivation curve shifted in the negative direction, half inactivation potential (V1/2) was -60.9±0.5mv (n=7) (p <0.05), slope factor K was -11.5±0.5 (p <0.05); Recovery time constantτfast andτslow were 2.1±0.2ms (n=6) ( p <0.05) and 42.5±6.9ms.Conclusions1. Nav1.1 E1623A displayed a decreased current density, a slower activation, a negative shift in the steady-state inactivation and a significantly delayed recovery from inactivation. These data indicates that Nav1.1 E1623A results in partial loss of function. 2. The E1623A missense mutation may decrease the availability of the Nav1.1 channel in inhibitory interneurons, resulting in the partial dysfunction of network inhibition. The resulting dysfunction of network inhibition can hyperactivate excitatory neurons leading to epilepsy.更多还原