自发性癫痫大鼠海马电压门控性钠通道的表达研究
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摘要
前言
电压门控性钠通道为一种跨膜的糖蛋白,其在神经元动作电位的产生与传播过程中发挥重要作用。在中枢神经系统中,钠通道通常由一个α型大亚基(260KD)和一个或多个β型小亚基(32～37KD)组成,其结构均由四个有50%序列同源的结构域(Ⅰ～Ⅳ)组成,每个结构域含有6个跨膜片段(S_1～S_6)。研究表明α型大亚基与β型小亚基均具有对钠通道失活与激活动力学的调节作用;此外β型小亚基可以作为细胞黏附因子来发挥调节作用。
哺乳动物的α型大亚基根据不同的组织特异性与生物物理学特性由9个基因编码。中枢神经系统中,Nav1.1、Nav1.2、Nav1.3和Nav1.6表达较多,其中Nav1.1在海马、小脑、脊髓、脑干等部位表达很高。已有钠通道的200多种基因突变在不同癫痫表型的研究中被证实。伴热惊厥性全身性癫痫(Generalized epilepsy withfebrile seizure plus,GEFS~+)是一种常染色体显性遗传的特发性全身性癫痫,SCN1A(编码Nav1.1的基因)和SCN1B(编码β_1的基因)的突变可引起GEFS~+。Nav1.3的mRNA在癫痫患者的海马CA4区表达显著升高;而对于癫痫持续状态诱发后4小时的新生鼠,Nav1.3的mRNA在其海马CA1、CA3区以及齿状回颗粒细胞区表达上调。因此,以往很多研究均表明电压门控性钠通道的遗传异常表达可能会引起中枢神经系统神经元的兴奋性变化。
自发性癫痫大鼠(Spontaneously epileptic rat,SER)是一种纯合型的双基因突变大鼠:其突变基因tm来自于东京Wistar大鼠突变系Tremor,突变基因zi来自于德国斯普拉格——道利鼠突变系Zitter,两种突变基因均为常染色体隐性遗传。出生后8周,SER自发性地出现癫痫大发作和失神样的小发作,其脑电图典型表现为皮质与海马区出现的5～7赫兹的棘慢复合波。到目前为止,SER被认为是研究人类癫痫最好的动物模型之一,抗癫痫药物对SER的治疗作用反应与对人类癫痫作用十分相近。有报道利用SER进行有关改善天冬氨酸酰酶的基因治疗;而涉及离子通道病方面,仅报道了SER电压门控性钙通道的功能变化。
然而,迄今为止,有关SER电压门控钠通道的研究仍为空白。在本研究中,我们假设SER在其遗传性癫痫发生发展中涉及到钠通道的参与与变化。因此,我们拟从基因与蛋白两个层面研究SER电压门控性钠通道的表达情况,以期明确其在癫痫海马兴奋性中的角色与作用。
实验材料和方法
1、材料
动物、试剂与仪器:健康SER和正常Wistar大鼠9～12周龄,每个实验组均为6只;Wistar新生鼠5只,均小于一周龄;雌雄不限,均由中国医科大学实验动物部提供。Trizol(GIBCO BRL),免疫组化SP试剂盒(北京中山公司),实时定量RT-PCR试剂盒(Takara),引物由上海生工生物工程公司合成,其他药物均为国产分析纯。Ⅰ型与Ⅲ型钠通道亚基兔多克隆抗体(anti-Nav1.1,anti-Nav1.3,Sigma),anti-β_(1ex)由密歇根大学Lori L.Isom博士赠送;CM1800 LEICA冰冻切片机(德国),PCR仪(PE-9600,PE-2400),凝胶自动成像仪(美国,GDSH型),MetaMor2ph/DP10/BX41图像分析系统(美国)。
2、方法
RT-PCR分析:总的RNA从SER与正常Wistar大鼠整个海马组织中提取,参照Takara试剂盒进行实时定量RT-PCR操作。目的基因Nav1.1、Nav1.3和β_1亚基mRNA的相对表达水平用目的基因与管家基因的平均光密度比值确定。
实时定量RT-PCR分析:总的RNA从SER与正常Wistar大鼠整个海马组织中提取,参照Takara试剂盒进行实时定量RT-PCR操作。目的基因七点标准曲线通过反转录产物五倍稀释物进行。目的基因Nav1.1、Nav1.3和β_1亚基mRNA的相对表达水平用目的基因与管家基因的拷贝数比值确定。
免疫荧光分析:SER组、正常对照组与新生鼠组(阳性对照)腹腔麻醉后快速取出鼠脑,常规制作冰冻切片。钠通道亚基兔多克隆抗体一抗(anti-Nav1.1,1:25稀释;anti-β_(1ex),1:100稀释;anti-Nav1.3,1:25稀释)常温30分钟后4℃过夜,用FITC标记的羊抗兔二抗孵育切片,PBS漂洗三次后,荧光显微镜观察。
免疫组化分析:SER组与正常组常规做石蜡切片。钠通道亚基兔多克隆一抗(anti-β_(1ex),1:100稀释;anti-Nav1.1,1:25稀释)孵育切片,常温30分钟后4℃过夜。用针对一抗的生物素标记的羊抗兔二抗孵育切片,37℃1小时。DAB显色后,用苏木精做核复染。每只大鼠取10张脑片,其断面水平各鼠均相似,所有脑片均在同一放大倍数(×400)、同一光强度下分析。首先分析海马内平均阳性细胞数,然后用MetaMorph/DP10/BX41图像分析系统测定各组海马CA1、CA3与齿状回蛋白阳性反应物的平均灰度值。
免疫印迹分析:SER组、正常对照组与新生鼠组(阳性对照)腹腔麻醉后快速取出鼠海马,提取总蛋白。考马斯亮蓝法测蛋白浓度,上样后进行SDS-PAGE蛋白电泳,一抗(anti-β_(1ex),1:500稀释;anti-Nav1.1,1:200稀释;anti-Nav1.3,1:200稀释)孵育。ECL与DAB显影。
统计学分析:各组结果以平均值±标准差表示,两组间比较用t检验。
结果
1、RT-PCR结果显示:SER海马Nav1.1 mRNA的表达显著高于正常对照组(1.14±0.16 in SERs vs 0.68±0.11 in control rats;p＜0.001);SER海马Nav1.3mRNA的表达亦显著高于正常对照组(0.89±0.17 in SERs vs 0.45±0.16 in controlrats;p＜0.001);同样的,SER海马β_1亚基mRNA的表达显著高于正常对照组(1.12±0.11 in SERs vs 0.91±0.14 in control rats;p＜0.05)。
2、实时定量RT-PCR结果显示:SER海马Nav1.1 mRNA的表达显著高于正常对照组(1.01±0.37 in SERs vs 0.36±0.11 in control rats;p＜0.001);SER海马Nav1.3mRNA的表达亦显著高于正常对照组(0.55±0.15 in SERs vs 0.39±0.15 in controlrats;p＜0.001);同样的,SER海马β_1亚基mRNA的表达显著高于正常对照组(0.62±0.13 in SERs vs 0.52±0.07 in control rats;p＜0.05)。综上,SER海马Nav1.1、Nav1.3和β_1亚基在mRNA水平表达上调。
3、免疫荧光结果显示:Nav1.1和β_1亚基蛋白在SER和正常大鼠海马各区均有表达;Nav1.3蛋白在新生鼠(三天龄,阳性对照)海马有表达;然而,在正常对照大鼠中未见显著表达Nav1.3蛋白的阳性细胞;值得注意的是,正常对照大鼠微量表达的Nav1.3蛋白在SER海马有显著的阳性表达。
4、免疫组化结果证实:Nav1.1和β_1亚基蛋白免疫反应性的阳性细胞在SER和正常大鼠海马各区均有表达,包括CA1、CA3以及齿状回颗粒细胞区;SER海马各区Nav1.1和β_1亚基蛋白免疫反应性的阳性细胞数和平均灰度值均显著高于正常对照大鼠,提示SER海马各区Nav1.1和β_1亚基蛋白表达上调。
5、免疫印迹结果进一步证实:SER海马Nav1.1蛋白表达显著高于正常对照组(0.52±0.05 in SERs vs 0.30±0.04 in controlrats;p＜0.001);SER海马Nav1.3蛋白表达亦显著高于正常对照组(0.38±0.07 in SERs vs 0.17±0.05 in control rats;p＜0.001);然而,SER海马Nav1.3蛋白表达与新生鼠阳性对照组无统计学差异(0.38±0.07 in SERs vs 0.37±0.05 in neonatal rats;p＞0.05);SER海马β_1亚基蛋白的表达亦显著高于正常对照组(0.40±0.06 in SERs vs 0.29±0.05 in control rats;p＜0.05)。综上,SER海马Nav1.1、Nav1.3和β_1亚基在蛋白水平表达上调。
结论
1、首次证实,与正常Wistar大鼠比较,SER海马Nav1.1、Nav1.3和β_1亚基在mRNA水平表达上调。
2、首次发现,Nav1.1和β_1蛋白在SER海马各区均有表达,包括CA1、CA3以及齿状回颗粒细胞区。
3、首次证实,与正常Wistar大鼠比较,SER海马Nav1.1和β_1亚基在蛋白水平表达上调。
4、首次发现,正常成熟的Wistar大鼠微量表达的Nav1.3蛋白在成熟的SER海马有显著的阳性表达。
5、SER海马Nav1.1、Nav1.3和β_1亚基在mRNA与蛋白水平表达上调,可能会促进癫痫样兴奋活动的产生,进而构成SER癫痫表型的发作基础。
6、SER作为一种良好的癫痫动物模型,可用其筛选钠通道特异性亚型与亚基药物并应用于基因治疗。
7、本研究结果在解释遗传性癫痫海马兴奋性的分子机制上有其重要意义。
Introduction
Voltage-gated sodium channels(VGSCs)are the primary molecules responsible for the rising phase of action potentials in electrically excitable cells,which consist of a complex of different glycosylated subunits,including a pore-forming a subunit of 260 kDa and fourβsubunits of 32-36 kDa(β_1-β_4 subunit).Theαsubunit forms the ion-selective pore,inner pore,voltage sensor and inactivation gate,including a transmembrane protein that consists of four homologous domains(Ⅰ-Ⅳ),each with six transmembrane segments.It is generally agreed that both a andβsubunits play critical roles in modulating the activation and inactivation kinetics of sodium current.In addition,β_1 subunit may function as cell adhesion molecules.
Nine mammalian VGSC isoforms have already been identified.In the central nervous system(CNS),Nav1.1,Nav1.2,Nav1.3 and Nav1.6 are abundantly expressed, and Nav1.1 has been recognized to be highly detectable in the hippocampus, cerebellum,spinal cord,brainstem,neo-cortex,substantia nigra and caudate.Until now, many mutations in VGSC genes have been shown to be critically linked to specific epileptic syndromes.In generalized epilepsy with febrile seizures plus(GEFS~+),an autosomal dominant epilepsy syndrome,mutations in the genes coding for VGSCⅠα-isoform(Nav1.1)or;β_1 subunit(SCN1A,SCN1B)have been demonstrated.In human epilepsy,VGSCⅠa-isoform(Nav1.3)mRNA was found to be expressed at significantly higher levels in CA4 hilar cells in the epileptic hippocampus.It has also been reported that increased expression of neonatal Nay 1.3 mRNA was observed 4h after the induction of status epilepticus in neurons of CA1-CA3 and the dentate granule cell layer.Therefore,many previous studies have suggested that genetic abnormalities in the VGSC isoforms predominantly are likely to contribute to neuronal excitability in the CNS.
The spontaneously epileptic rat(SER)is a double mutant obtained by mating heterozygous tremor rats(tm)(tm/+)and homozygous zitter rats(zi)(zi/zi).SER exhibits spontaneous tonic convulsions and absence-like seizures,characterized by simultaneous appearance of 5-7Hz spike-wave complexes in cortical and hippocampal EEG after the age of 8 weeks.The profiles of conventional antiepileptic drugs in SER are quite similar to the efficacy profile in human epilepsy.The mechanism underlying the epileptic seizures in SER was thought to include an abnormality of Ca channel function,an increase in extracellular glutamate concentrations,and enhanced levels of TVacetylaspartate because of lack of the aspartoacylase gene.
However,so far,the effects of VGSC in SER have not yet been elucidated.We hypothesized that the etiopathogenisis of SER in genetic epilepsy might be involved in changes of VGSC.Thus,we investigated the expressions of VGSC in SER by researching VGSC Nav1.1,Nay1.3 andβ_1 subunit at the mRNA and protein expression levels of SER,in hope that an insight in their roles in hippocampal excitability could be obtained.
Materials and Methods
1.Materials
Normal Wistar rats and SERs at the age of 9-12 weeks were housed in individual cages under a controlled environment(12:12 h light/dark cycle,50-70%humidity, 24℃).Food and water were available ad libitum.For each experimental method,six SERs were in each experimental group and six Wistar rats were in each control group.
The VGSCβ_1 polyclonal antibody generated against the extracellular domain of VGSCβ_1 subunit(KRRSETTAETFTEWTFR),was a gift from Doctor L.L.Isom, synthesized by the Protein and Carbohydrate Structure Core facility at the University of Michigan.The Nav1.1 and Nav1.3 antibodies were purchased from Sigma.
2.Methods
RT-PCR analysis:Total RNA was prepared from the whole hippocampus tissues of SERs(n=6)and control Wistar rats(n=6)using TRIzol(Life Technologies) according to manufacturer's instructions(Takara).
Real-time RT-PCR analysis:Total RNA was prepared from the whole hippocampus tissues of SERs(n=6)and control Wistar rats(n=6)using TRIzol(Life Technologies)according to manufacturer's instructions(Takara).A seven point standard curve for each gene was constructed using five fold serial dilutions of RT product.
Immunofluorescence:The brains of SERs(n=6)and Wistar rats(n=6)were dissected out under anesthesia.Neonatal rat hippocampus was tested for Nav1.3 protein analysis and served as a positive control.Tissue sections were incubated with the primary antibody(anti-Nav1.1,1:25 dilution;anti-β_(1ex),1:100 dilution;anti-Nav1.3, 1:25 dilution).The sections were incubated at 30 min room temperature and overnight at 4℃,followed by incubation for 30 min in FITC-labeled goat anti-rabbit secondary antibody,and cell nuclei were labeled with Hoechst33258.After washes in PBS,the sections were mounted,viewed and photographed utilizing fluorescence microscopy.
Immunohistochemistry:SERs(n=6)and Wistar rats(n=6)were perfused intracardially under anesthesia with normal saline,followed by 4%paraformaldehyde ice-cold fixative.Brains were dissected out and placed in 4%paraformaldehyde for 20 h at 4℃.The primary antibodies(anti-β_(1ex),1:100 dilution;anti-Nav1.1,1:25 dilution) were incubated for 30 min at room temperature and overnight at 4℃,and they were carried out using avidin-biotin peroxidase method and 3,3′-diaminobenzidine(DAB) as a chromogen.Control sections incubated without the primary antibody or with pre-immune sera were performed.The intensity,the cellular localization,and the frequency of immunoreactive cells were examined in different hippocampal regions (CA1,CA3 and dentate gyrus)of control rats and SERs.The expression levels of Nav1.1 andβ_1 subunit were quantified by counting immunoreactive cells and measuring the intensity of staining using a computerized image analysis system.
Western blot analysis:Western blot analysis was performed on samples of the whole hippocampus of adult SERs(n=6)and adult Wistar rats(n=6).For Nav1.3 protein analysis,six neonatal rats were served as positive controls.Samples were homogenized in lysis buffer containing 10 mM Tris(pH 8.0),150 mM NaCl,10% glycerol,1%NP-40,5 mM ethylenediamine tetra-acetic acid(EDTA)and protease inhibitor cocktail.Samples were incubated over night in TTBS:3%BSA,0.1%sodium azide,containing the primary antibodies(anti-β_(1ex),1:500 dilution;anti-Nav1.1,1:200 dilution;anti-Nav1.3,1:200 dilution).Immunoreactive bands were visualized using DAB and ECL kits.The levels of Nav1.1,Nav1.3 andβ_1 protein were evaluated by measuring optical densities of the protein bands using Scion Image for Windows image-analysis software.
Statistical analyses:Mean and standard deviation were calculated for all measurements in this study.Student's t-test was performed to determine statistical significance,which was evaluated at p＜0.05.All statistical analyses were conducted using SPSS for Windows version 12.0.
Results
1.The mRNA expressions of VGSC Nav1.1,Nav1.3 andβ_1 subunit in SERs and control rats hippocampus
The data suggested that the mRNA expression of Nav1.1 in SERs hippocampus was significantly higher than that of control groups(1.01±0.37 in SERs vs 0.36±0.11 in control rats;p＜0.001)by real-time RT-PCR;The mRNA expression level of Nav1.3 was abundantly increased compared with the control rats(0.55±0.15 in SERs vs 0.39±0.15 in control rats;p＜0.001);The mRNA expression ofβ_1 subunit was also higher than that of control groups in hippocampus(0.62±0.13 in SERs vs 0.52±0.07 in control rats;p＜0.05).The data by real-time RT-PCR were in agreement with the data by means of RT-PCR.On the whole,the mRNA expressions of VGSC Nav1.1,Nav1.3 andβ_1 subunit were up-regulated in SERs hippocampus.
2.The localization expressions of VGSC Nav1.1,Nav1.3 andβ_1 subunit in SERs and control rats hippocampus
Nav1.1 andβ_1 protein in SERs and control tissue were localized widely in the hippocampus by immunofluorescence.Under our investigation,Nav1.3 protein that was expressed in the neonatal(at the age of 3 days)hippocampus,was considered as a positive control.It was noting that the clear Nav1.3-immunoreactive cells were not found in control adult Wistar rats hippocampus.However,Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.
3.The protein expressions of VGSC Nav1.1 andβ_1 subunit in SERs and control rats hippocampus
By means of immunohistochemistry,the protein expressions of Nav1.1 andβ_1 subunit in the hippocampus of SERs were significantly higher than those in control rats. In addition,the increases in Nav1.1 andβ_1 staining were clearly present throughout the hippocampus of SERs,including CA1,CA3 and dentate gyrus regions.Taken together, the data showed that the expressions at the protein level of Nav1.1 andβ_1 subunit were up-regulated in SERs hippocampus.
4.Additional tests that support the protein expressions of VGSC Nav1.1,Nav1.3 andβ_1 subunit in SERs and control rats hippocampus
The protein expression of Nav1.1 in SERs hippocampus was significantly higher than that in control groups by western blot analysis(0.52±0.05 in SERs vs 0.30±0.04 in control rats;p＜0.001);The protein expression level of Nav1.3 was abundantly increased compared with the control rats(0.38±0.07 in SERs vs 0.17±0.05 in control rats;p＜0.001).However,it seemed unchanged compared with neonatal rats(0.38±0.07 in SERs vs 0.37±0.05 in neonatal rats;p＞0.05).Taken together with the data of immunofluorescence,Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.The protein expression ofβ_1 subunit in SERs was higher than that of control rats in hippocampus(0.40±0.06 in SERs vs 0.29±0.05 in control rats;p＜0.05).Altogether,the findings using western blot analysis further verified up-regulation expressions of VGSC Nav1.1,Nav1.3 andβ_1 subunit at the protein level in SERs hippocampus.
Conclusions
1.The mRNA expressions of Nav1.1,Nav1.3 andβ_1 subunit were first detected to be up-regulated in SERs hippocampus compared with control Wistar rats.
2.Nav1.1 andβ_1 subunit were first observed to be localized widely in SER hippocampus including CA1-CA3 and DG.
3.The protein expressions of Nav1.1 andβ_1 subunit were first found to be up-regulated in SERs hippocampus compared with control Wistar rats.
4.Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.
5.Sodium channel Nav1.1,Nav1.3 andβ_1 subunit up-regulation at the mRNA and protein levels of SER hippocampus might contribute to the generation of epileptiform activity and underlie the observed seizure phenotype in SER.
6.The SER can be used as an epileptic animal model to screen effective specific VGSC isoforms for medicine and gene therapy.
7.The results of this study may be of value in revealing components of the molecular mechanisms of hippocampal excitation that are related to genetic epilepsy.
电压门控性钠通道为一种跨膜的糖蛋白,其在神经元动作电位的产生与传播过程中发挥重要作用。在中枢神经系统中,钠通道通常由一个α型大亚基(260KD)和一个或多个β型小亚基(32～37KD)组成,其结构均由四个有50%序列同源的结构域(Ⅰ～Ⅳ)组成,每个结构域含有6个跨膜片段(S_1～S_6)。研究表明α型大亚基与β型小亚基均具有对钠通道失活与激活动力学的调节作用;此外β型小亚基可以作为细胞黏附因子来发挥调节作用。
哺乳动物的α型大亚基根据不同的组织特异性与生物物理学特性由9个基因编码。中枢神经系统中,Nav1.1、Nav1.2、Nav1.3和Nav1.6表达较多,其中Nav1.1在海马、小脑、脊髓、脑干等部位表达很高。已有钠通道的200多种基因突变在不同癫痫表型的研究中被证实。伴热惊厥性全身性癫痫(Generalized epilepsy withfebrile seizure plus,GEFS~+)是一种常染色体显性遗传的特发性全身性癫痫,SCN1A(编码Nav1.1的基因)和SCN1B(编码β_1的基因)的突变可引起GEFS~+。Nav1.3的mRNA在癫痫患者的海马CA4区表达显著升高;而对于癫痫持续状态诱发后4小时的新生鼠,Nav1.3的mRNA在其海马CA1、CA3区以及齿状回颗粒细胞区表达上调。因此,以往很多研究均表明电压门控性钠通道的遗传异常表达可能会引起中枢神经系统神经元的兴奋性变化。
自发性癫痫大鼠(Spontaneously epileptic rat,SER)是一种纯合型的双基因突变大鼠:其突变基因tm来自于东京Wistar大鼠突变系Tremor,突变基因zi来自于德国斯普拉格——道利鼠突变系Zitter,两种突变基因均为常染色体隐性遗传。出生后8周,SER自发性地出现癫痫大发作和失神样的小发作,其脑电图典型表现为皮质与海马区出现的5～7赫兹的棘慢复合波。到目前为止,SER被认为是研究人类癫痫最好的动物模型之一,抗癫痫药物对SER的治疗作用反应与对人类癫痫作用十分相近。有报道利用SER进行有关改善天冬氨酸酰酶的基因治疗;而涉及离子通道病方面,仅报道了SER电压门控性钙通道的功能变化。
然而,迄今为止,有关SER电压门控钠通道的研究仍为空白。在本研究中,我们假设SER在其遗传性癫痫发生发展中涉及到钠通道的参与与变化。因此,我们拟从基因与蛋白两个层面研究SER电压门控性钠通道的表达情况,以期明确其在癫痫海马兴奋性中的角色与作用。
实验材料和方法
1、材料
动物、试剂与仪器:健康SER和正常Wistar大鼠9～12周龄,每个实验组均为6只;Wistar新生鼠5只,均小于一周龄;雌雄不限,均由中国医科大学实验动物部提供。Trizol(GIBCO BRL),免疫组化SP试剂盒(北京中山公司),实时定量RT-PCR试剂盒(Takara),引物由上海生工生物工程公司合成,其他药物均为国产分析纯。Ⅰ型与Ⅲ型钠通道亚基兔多克隆抗体(anti-Nav1.1,anti-Nav1.3,Sigma),anti-β_(1ex)由密歇根大学Lori L.Isom博士赠送;CM1800 LEICA冰冻切片机(德国),PCR仪(PE-9600,PE-2400),凝胶自动成像仪(美国,GDSH型),MetaMor2ph/DP10/BX41图像分析系统(美国)。
2、方法
RT-PCR分析:总的RNA从SER与正常Wistar大鼠整个海马组织中提取,参照Takara试剂盒进行实时定量RT-PCR操作。目的基因Nav1.1、Nav1.3和β_1亚基mRNA的相对表达水平用目的基因与管家基因的平均光密度比值确定。
实时定量RT-PCR分析:总的RNA从SER与正常Wistar大鼠整个海马组织中提取,参照Takara试剂盒进行实时定量RT-PCR操作。目的基因七点标准曲线通过反转录产物五倍稀释物进行。目的基因Nav1.1、Nav1.3和β_1亚基mRNA的相对表达水平用目的基因与管家基因的拷贝数比值确定。
免疫荧光分析:SER组、正常对照组与新生鼠组(阳性对照)腹腔麻醉后快速取出鼠脑,常规制作冰冻切片。钠通道亚基兔多克隆抗体一抗(anti-Nav1.1,1:25稀释;anti-β_(1ex),1:100稀释;anti-Nav1.3,1:25稀释)常温30分钟后4℃过夜,用FITC标记的羊抗兔二抗孵育切片,PBS漂洗三次后,荧光显微镜观察。
免疫组化分析:SER组与正常组常规做石蜡切片。钠通道亚基兔多克隆一抗(anti-β_(1ex),1:100稀释;anti-Nav1.1,1:25稀释)孵育切片,常温30分钟后4℃过夜。用针对一抗的生物素标记的羊抗兔二抗孵育切片,37℃1小时。DAB显色后,用苏木精做核复染。每只大鼠取10张脑片,其断面水平各鼠均相似,所有脑片均在同一放大倍数(×400)、同一光强度下分析。首先分析海马内平均阳性细胞数,然后用MetaMorph/DP10/BX41图像分析系统测定各组海马CA1、CA3与齿状回蛋白阳性反应物的平均灰度值。
免疫印迹分析:SER组、正常对照组与新生鼠组(阳性对照)腹腔麻醉后快速取出鼠海马,提取总蛋白。考马斯亮蓝法测蛋白浓度,上样后进行SDS-PAGE蛋白电泳,一抗(anti-β_(1ex),1:500稀释;anti-Nav1.1,1:200稀释;anti-Nav1.3,1:200稀释)孵育。ECL与DAB显影。
统计学分析:各组结果以平均值±标准差表示,两组间比较用t检验。
结果
1、RT-PCR结果显示:SER海马Nav1.1 mRNA的表达显著高于正常对照组(1.14±0.16 in SERs vs 0.68±0.11 in control rats;p＜0.001);SER海马Nav1.3mRNA的表达亦显著高于正常对照组(0.89±0.17 in SERs vs 0.45±0.16 in controlrats;p＜0.001);同样的,SER海马β_1亚基mRNA的表达显著高于正常对照组(1.12±0.11 in SERs vs 0.91±0.14 in control rats;p＜0.05)。
2、实时定量RT-PCR结果显示:SER海马Nav1.1 mRNA的表达显著高于正常对照组(1.01±0.37 in SERs vs 0.36±0.11 in control rats;p＜0.001);SER海马Nav1.3mRNA的表达亦显著高于正常对照组(0.55±0.15 in SERs vs 0.39±0.15 in controlrats;p＜0.001);同样的,SER海马β_1亚基mRNA的表达显著高于正常对照组(0.62±0.13 in SERs vs 0.52±0.07 in control rats;p＜0.05)。综上,SER海马Nav1.1、Nav1.3和β_1亚基在mRNA水平表达上调。
3、免疫荧光结果显示:Nav1.1和β_1亚基蛋白在SER和正常大鼠海马各区均有表达;Nav1.3蛋白在新生鼠(三天龄,阳性对照)海马有表达;然而,在正常对照大鼠中未见显著表达Nav1.3蛋白的阳性细胞;值得注意的是,正常对照大鼠微量表达的Nav1.3蛋白在SER海马有显著的阳性表达。
4、免疫组化结果证实:Nav1.1和β_1亚基蛋白免疫反应性的阳性细胞在SER和正常大鼠海马各区均有表达,包括CA1、CA3以及齿状回颗粒细胞区;SER海马各区Nav1.1和β_1亚基蛋白免疫反应性的阳性细胞数和平均灰度值均显著高于正常对照大鼠,提示SER海马各区Nav1.1和β_1亚基蛋白表达上调。
5、免疫印迹结果进一步证实:SER海马Nav1.1蛋白表达显著高于正常对照组(0.52±0.05 in SERs vs 0.30±0.04 in controlrats;p＜0.001);SER海马Nav1.3蛋白表达亦显著高于正常对照组(0.38±0.07 in SERs vs 0.17±0.05 in control rats;p＜0.001);然而,SER海马Nav1.3蛋白表达与新生鼠阳性对照组无统计学差异(0.38±0.07 in SERs vs 0.37±0.05 in neonatal rats;p＞0.05);SER海马β_1亚基蛋白的表达亦显著高于正常对照组(0.40±0.06 in SERs vs 0.29±0.05 in control rats;p＜0.05)。综上,SER海马Nav1.1、Nav1.3和β_1亚基在蛋白水平表达上调。
结论
1、首次证实,与正常Wistar大鼠比较,SER海马Nav1.1、Nav1.3和β_1亚基在mRNA水平表达上调。
2、首次发现,Nav1.1和β_1蛋白在SER海马各区均有表达,包括CA1、CA3以及齿状回颗粒细胞区。
3、首次证实,与正常Wistar大鼠比较,SER海马Nav1.1和β_1亚基在蛋白水平表达上调。
4、首次发现,正常成熟的Wistar大鼠微量表达的Nav1.3蛋白在成熟的SER海马有显著的阳性表达。
5、SER海马Nav1.1、Nav1.3和β_1亚基在mRNA与蛋白水平表达上调,可能会促进癫痫样兴奋活动的产生,进而构成SER癫痫表型的发作基础。
6、SER作为一种良好的癫痫动物模型,可用其筛选钠通道特异性亚型与亚基药物并应用于基因治疗。
7、本研究结果在解释遗传性癫痫海马兴奋性的分子机制上有其重要意义。
Introduction
Voltage-gated sodium channels(VGSCs)are the primary molecules responsible for the rising phase of action potentials in electrically excitable cells,which consist of a complex of different glycosylated subunits,including a pore-forming a subunit of 260 kDa and fourβsubunits of 32-36 kDa(β_1-β_4 subunit).Theαsubunit forms the ion-selective pore,inner pore,voltage sensor and inactivation gate,including a transmembrane protein that consists of four homologous domains(Ⅰ-Ⅳ),each with six transmembrane segments.It is generally agreed that both a andβsubunits play critical roles in modulating the activation and inactivation kinetics of sodium current.In addition,β_1 subunit may function as cell adhesion molecules.
Nine mammalian VGSC isoforms have already been identified.In the central nervous system(CNS),Nav1.1,Nav1.2,Nav1.3 and Nav1.6 are abundantly expressed, and Nav1.1 has been recognized to be highly detectable in the hippocampus, cerebellum,spinal cord,brainstem,neo-cortex,substantia nigra and caudate.Until now, many mutations in VGSC genes have been shown to be critically linked to specific epileptic syndromes.In generalized epilepsy with febrile seizures plus(GEFS~+),an autosomal dominant epilepsy syndrome,mutations in the genes coding for VGSCⅠα-isoform(Nav1.1)or;β_1 subunit(SCN1A,SCN1B)have been demonstrated.In human epilepsy,VGSCⅠa-isoform(Nav1.3)mRNA was found to be expressed at significantly higher levels in CA4 hilar cells in the epileptic hippocampus.It has also been reported that increased expression of neonatal Nay 1.3 mRNA was observed 4h after the induction of status epilepticus in neurons of CA1-CA3 and the dentate granule cell layer.Therefore,many previous studies have suggested that genetic abnormalities in the VGSC isoforms predominantly are likely to contribute to neuronal excitability in the CNS.
The spontaneously epileptic rat(SER)is a double mutant obtained by mating heterozygous tremor rats(tm)(tm/+)and homozygous zitter rats(zi)(zi/zi).SER exhibits spontaneous tonic convulsions and absence-like seizures,characterized by simultaneous appearance of 5-7Hz spike-wave complexes in cortical and hippocampal EEG after the age of 8 weeks.The profiles of conventional antiepileptic drugs in SER are quite similar to the efficacy profile in human epilepsy.The mechanism underlying the epileptic seizures in SER was thought to include an abnormality of Ca channel function,an increase in extracellular glutamate concentrations,and enhanced levels of TVacetylaspartate because of lack of the aspartoacylase gene.
However,so far,the effects of VGSC in SER have not yet been elucidated.We hypothesized that the etiopathogenisis of SER in genetic epilepsy might be involved in changes of VGSC.Thus,we investigated the expressions of VGSC in SER by researching VGSC Nav1.1,Nay1.3 andβ_1 subunit at the mRNA and protein expression levels of SER,in hope that an insight in their roles in hippocampal excitability could be obtained.
Materials and Methods
1.Materials
Normal Wistar rats and SERs at the age of 9-12 weeks were housed in individual cages under a controlled environment(12:12 h light/dark cycle,50-70%humidity, 24℃).Food and water were available ad libitum.For each experimental method,six SERs were in each experimental group and six Wistar rats were in each control group.
The VGSCβ_1 polyclonal antibody generated against the extracellular domain of VGSCβ_1 subunit(KRRSETTAETFTEWTFR),was a gift from Doctor L.L.Isom, synthesized by the Protein and Carbohydrate Structure Core facility at the University of Michigan.The Nav1.1 and Nav1.3 antibodies were purchased from Sigma.
2.Methods
RT-PCR analysis:Total RNA was prepared from the whole hippocampus tissues of SERs(n=6)and control Wistar rats(n=6)using TRIzol(Life Technologies) according to manufacturer's instructions(Takara).
Real-time RT-PCR analysis:Total RNA was prepared from the whole hippocampus tissues of SERs(n=6)and control Wistar rats(n=6)using TRIzol(Life Technologies)according to manufacturer's instructions(Takara).A seven point standard curve for each gene was constructed using five fold serial dilutions of RT product.
Immunofluorescence:The brains of SERs(n=6)and Wistar rats(n=6)were dissected out under anesthesia.Neonatal rat hippocampus was tested for Nav1.3 protein analysis and served as a positive control.Tissue sections were incubated with the primary antibody(anti-Nav1.1,1:25 dilution;anti-β_(1ex),1:100 dilution;anti-Nav1.3, 1:25 dilution).The sections were incubated at 30 min room temperature and overnight at 4℃,followed by incubation for 30 min in FITC-labeled goat anti-rabbit secondary antibody,and cell nuclei were labeled with Hoechst33258.After washes in PBS,the sections were mounted,viewed and photographed utilizing fluorescence microscopy.
Immunohistochemistry:SERs(n=6)and Wistar rats(n=6)were perfused intracardially under anesthesia with normal saline,followed by 4%paraformaldehyde ice-cold fixative.Brains were dissected out and placed in 4%paraformaldehyde for 20 h at 4℃.The primary antibodies(anti-β_(1ex),1:100 dilution;anti-Nav1.1,1:25 dilution) were incubated for 30 min at room temperature and overnight at 4℃,and they were carried out using avidin-biotin peroxidase method and 3,3′-diaminobenzidine(DAB) as a chromogen.Control sections incubated without the primary antibody or with pre-immune sera were performed.The intensity,the cellular localization,and the frequency of immunoreactive cells were examined in different hippocampal regions (CA1,CA3 and dentate gyrus)of control rats and SERs.The expression levels of Nav1.1 andβ_1 subunit were quantified by counting immunoreactive cells and measuring the intensity of staining using a computerized image analysis system.
Western blot analysis:Western blot analysis was performed on samples of the whole hippocampus of adult SERs(n=6)and adult Wistar rats(n=6).For Nav1.3 protein analysis,six neonatal rats were served as positive controls.Samples were homogenized in lysis buffer containing 10 mM Tris(pH 8.0),150 mM NaCl,10% glycerol,1%NP-40,5 mM ethylenediamine tetra-acetic acid(EDTA)and protease inhibitor cocktail.Samples were incubated over night in TTBS:3%BSA,0.1%sodium azide,containing the primary antibodies(anti-β_(1ex),1:500 dilution;anti-Nav1.1,1:200 dilution;anti-Nav1.3,1:200 dilution).Immunoreactive bands were visualized using DAB and ECL kits.The levels of Nav1.1,Nav1.3 andβ_1 protein were evaluated by measuring optical densities of the protein bands using Scion Image for Windows image-analysis software.
Statistical analyses:Mean and standard deviation were calculated for all measurements in this study.Student's t-test was performed to determine statistical significance,which was evaluated at p＜0.05.All statistical analyses were conducted using SPSS for Windows version 12.0.
Results
1.The mRNA expressions of VGSC Nav1.1,Nav1.3 andβ_1 subunit in SERs and control rats hippocampus
The data suggested that the mRNA expression of Nav1.1 in SERs hippocampus was significantly higher than that of control groups(1.01±0.37 in SERs vs 0.36±0.11 in control rats;p＜0.001)by real-time RT-PCR;The mRNA expression level of Nav1.3 was abundantly increased compared with the control rats(0.55±0.15 in SERs vs 0.39±0.15 in control rats;p＜0.001);The mRNA expression ofβ_1 subunit was also higher than that of control groups in hippocampus(0.62±0.13 in SERs vs 0.52±0.07 in control rats;p＜0.05).The data by real-time RT-PCR were in agreement with the data by means of RT-PCR.On the whole,the mRNA expressions of VGSC Nav1.1,Nav1.3 andβ_1 subunit were up-regulated in SERs hippocampus.
2.The localization expressions of VGSC Nav1.1,Nav1.3 andβ_1 subunit in SERs and control rats hippocampus
Nav1.1 andβ_1 protein in SERs and control tissue were localized widely in the hippocampus by immunofluorescence.Under our investigation,Nav1.3 protein that was expressed in the neonatal(at the age of 3 days)hippocampus,was considered as a positive control.It was noting that the clear Nav1.3-immunoreactive cells were not found in control adult Wistar rats hippocampus.However,Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.
3.The protein expressions of VGSC Nav1.1 andβ_1 subunit in SERs and control rats hippocampus
By means of immunohistochemistry,the protein expressions of Nav1.1 andβ_1 subunit in the hippocampus of SERs were significantly higher than those in control rats. In addition,the increases in Nav1.1 andβ_1 staining were clearly present throughout the hippocampus of SERs,including CA1,CA3 and dentate gyrus regions.Taken together, the data showed that the expressions at the protein level of Nav1.1 andβ_1 subunit were up-regulated in SERs hippocampus.
4.Additional tests that support the protein expressions of VGSC Nav1.1,Nav1.3 andβ_1 subunit in SERs and control rats hippocampus
The protein expression of Nav1.1 in SERs hippocampus was significantly higher than that in control groups by western blot analysis(0.52±0.05 in SERs vs 0.30±0.04 in control rats;p＜0.001);The protein expression level of Nav1.3 was abundantly increased compared with the control rats(0.38±0.07 in SERs vs 0.17±0.05 in control rats;p＜0.001).However,it seemed unchanged compared with neonatal rats(0.38±0.07 in SERs vs 0.37±0.05 in neonatal rats;p＞0.05).Taken together with the data of immunofluorescence,Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.The protein expression ofβ_1 subunit in SERs was higher than that of control rats in hippocampus(0.40±0.06 in SERs vs 0.29±0.05 in control rats;p＜0.05).Altogether,the findings using western blot analysis further verified up-regulation expressions of VGSC Nav1.1,Nav1.3 andβ_1 subunit at the protein level in SERs hippocampus.
Conclusions
1.The mRNA expressions of Nav1.1,Nav1.3 andβ_1 subunit were first detected to be up-regulated in SERs hippocampus compared with control Wistar rats.
2.Nav1.1 andβ_1 subunit were first observed to be localized widely in SER hippocampus including CA1-CA3 and DG.
3.The protein expressions of Nav1.1 andβ_1 subunit were first found to be up-regulated in SERs hippocampus compared with control Wistar rats.
4.Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.
5.Sodium channel Nav1.1,Nav1.3 andβ_1 subunit up-regulation at the mRNA and protein levels of SER hippocampus might contribute to the generation of epileptiform activity and underlie the observed seizure phenotype in SER.
6.The SER can be used as an epileptic animal model to screen effective specific VGSC isoforms for medicine and gene therapy.
7.The results of this study may be of value in revealing components of the molecular mechanisms of hippocampal excitation that are related to genetic epilepsy.
引文
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24 Akopian AN,Souslova V,England S,et al.The tetrodotoxin-resistent sodium channel SNS has a specialized function in pain pathways.Nature Neuroscience.1999;2:541-548.
25 Chen J,Sochivko D,Beck H,et al.Activity-induced expression of common reference genes in individual ens neurons.Lab Invest.2001;81:913-916.
26 Ellerkmann RK,Remy S,Chen J,etal.Molecular and functional changes in voltage-dependent Na(+)channels following pilocarpine-induced status epilepticus in rat dentate granule cells.Neuroscience.2003;119:323-333.
27 Kalume F,Yu FH,Westenbroek,et al.Reduced sodium current in Purkinje neurons from Nay 1.1 mutant mice:implications for ataxia in severe myoclonic epilepsy in infancy.J Neurosci.2007;27:11065-11074.
28 Klein JP,Khera DS,Nersesyan H,et al.Dysregulation of sodium channel expression in cortical neurons in a rodent model of absence epilepsy,Brain Res.2004;1000:102-109.
29 Whitaker WR,Faull RL,Dragunow M,et al.Changes in the mRNAs encoding voltage-gated sodium channel type Ⅱ and Ⅲ in human epileptic hippocampus,Neuroscience.2001;106:275-285.
30 Aronica E,Yankaya B,Troost D,et al.Induction of neonatal sodium channel Ⅱ and Ⅲ a-isoform mRNAs in neurons and microglia after status epilepticus in the rat hippocampus,Eur J Neurosci.2001;13:1261-1266.
31 Copley RR,Evolutionary convergence of alternative splicing in ion channels.Trends Genet.2004;20:171-176.
32 Felts PA,Yokoyama S,Dib-Hajj S,et al.Sodium channel alpha-subunit mRNAs Ⅰ,Ⅱ,Ⅲ,NaG,Na6 and hNE (PN1):different expression patterns in developing rat nervous system.Mol Brain Res.1997;45:71-82.
33 Goldin AL.Evolution of voltage-gated Na~+ channels.J Exp Biol.2002;205:575-584.
34 Aronica E,Yankaya B,Troost D,et al.Induction of neonatal sodium channel Ⅱ and Ⅲ a-isoform mRNAs in neurons and microglia after status epilepticus in the rat hippocampus.Eur J Neurosci.2001;13:1261-1266.
35 Yu FH,Mantegazza M,Westenbroek RE,et al.Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy.Nat Neurosci.2006;9: 1142-1149.
36 Ito M,Shirasaka Y,Hirose S,et al.Seizure phenotypes of a family with missense mutations in SCN2A.Pediatr Neurol.2004;31:150-152.
37 Striano,Bordo L,Lispi ML,et al.A novel SCN2A mutation in family with benign familial infantile seizures.Epilepsia.2006;47:218-220.
38 Misra SN,Kahlig KM,George AL Jr.Impaired Na_v1.2 function and reduced cell surface expression in benign familial neonatal-infantile seizures.Epilepsia.2008;49:1535-1545.
39 Scalmani P,Rusconi R,Armatura E,et al.Effects in neocortical neurons of mutations of the Na(v)1.2 Na~+ channel causing benign familial neonatal-infantile seizures.J Neurosci.2006;26:10100-10109.
40 Xu R,Thomas EA,Jenkins M,et al.A childhood epilepsy mutation reveals a role for developmentally regulated splicing of a sodium channel.Mol Cell Neurosci.2007;35:292-301.
41 Morgan K,Stevens EB,Shah B,et al.Beta 3:an additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics.Proc Natl Acad Sci USA.2000;97:2308-2313.
42 Johnson D,Montpetit ML,Stocker PJ,et al.The sialic acid component of the β_1 subunit modulates voltage-gated sodium channel function.J Biol Chem.2004;279:44303-44310.
43 Dyke PM,Laurence SM,Chen CL,et al.Sodium Channel β_1 subunit mediated modulation of Na_v1.2 currents and cell surface density is dependent on interactions with contactin and ankyrin.J Biol Chem.2004;279:16044-16049.
44 Malhotra JD,Kazen-Gillespie K,Hortsch M,et al.Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact.J Biol Chem.2000;275:11383-11388.
45 Makita N,Sloan-Brown K,Weghuis DO,et al.Genomic organization and chromosomal assignment of the human voltage-gated Na(+)channel beta-1 subunit gene (SCN1B).Genomics.1994;23:628-634.
46 Isom LL.Sodium channel beta subunits:anything but auxiliary.Neuroscientist.2001;7:42-54.
47 Wallace RH,Wang DW,Singh R,et al.Febrile seizures and generalized epilepsy associated with a mutation in the Na~+ channel β_1 subunit gene SCN1B.Nat Genet.1998;19:366-370.
48 Wallace RH,Scheffer IE,Parasivam G,et al.Generalized epilepsy with febrile seizures plus:mutation of the sodium channel subunit SCN1B.Neurology.2002;58:1426-1429.
49 Scheffer IE,Harkin LA,Grinton BE,et al.Temporal lobe epilepsy and GEFS+ phenotypes associated with SCN1B mutations.Brain.2007;130:100-109.
50 Fjelll J,Dib-Hajj S,Fried K,et al.Differential expression of sodium channel genes in retinal ganglion cells.Brain Res Mol Brain Res.1997;15:197-204.
51 Moran O,Conti F.Skeletal muscle sodium channel is affected by an epileptogenic β_1 subunit mutation.Biochem.2001;282:55-59.
52 Meadows LS,Malhotra J,Loukas A,et al.Functional and biochemical analysis of a sodium channel β_1 subunit mutation responsible for generalized epilepsy with febrile seizures plus type 1.Neuroscience.2002;22:10699-10709.
53 Gorter JA,van Vliet EA,Lopes da Silva FH,et al.Sodium channel p,subunit expression is increased in reactive astrocytes in a rat model for mesial temporal lobe epilepsy.Eur J Neurosci.2002;16:360-364.
54 Chen CL,Ruth E,Xu XR,et al.Mice lacking sodium channel β_1 subunits display defects in neuronal excitability,sodium channel expression and nodal architecture.Neuroscience.2004;24:4030-4042.
1 K(o|¨)hling R.Voltage-gated sodium channels in epilepsy.Epilepsia.2002;43:1278-1295.
2 Hirose S,Okada M,Kaneko S,et al.Are some idiopathic epilepsies disorders of ion channels? A working hypothesis.Epilepsy Research.2000;41:191-194.
3 Catterall WA.Molecular properties of brain sodium channels:an important target for anticonvulsants.Adv Neurology.1999;79:441-456.
4 Johnson D,Montpetit ML,Stocker PJ,et al.The sialic acid component of the β_1 subunit modulates voltage-gated sodium channel function.J Biol Chem.2004;279:44303-44310.
5 Meisler MH and Kearney JA.Sodium channel mutations in epilepsy and other neurological disorders.J Clin Invest.2005;115:2010-2017.
6 Malhotra JD,Kazen-Gillespie K,Hortsch M,et al.Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact.J Biol Chem.2000;275:11383-11388.
7 Makita N,Sloan-Brown K,Weghuis DO,et al.Genomic organization and chromosomal assignment of the human voltage-gated Na(+)channel beta-1 subunit gene (SCN1B).Genomics.1994;23:628-634.
8 Morgan K,Stevens EB,Shah B,et al.Beta 3:an additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics.Proc Natl Acad Sci USA.2000;97:2308-2313.
9 Isom LL.Sodium Channel p Subunits:Anything but Auxiliary.Neuroscientist.2001;7:42-54.
10 Chen CL,Ruth E,Xu XR,et al.Mice Lacking Sodium Channel β_1 Subunits Display Defects in Neuronal Excitability,Sodium Channel Expression,and Nodal Architecture.Neuroscience.2004;24:4030-4042.
11 Meadows LS,Chen YH,Powell AJ,et al.Functional modulation of human brain Navl.3 sodium channels,expressed in mammalian cells,by auxiliary beta 1,beta 2 and beta 3 subunits.Neuroscience.2002;114:745-753.
12 Johnson D,Montpetit ML,Stocker PJ,et al.The Sialic Acid Component of the β_1 Subunit Modulates Voltage-gated Sodium Channel Function.J Biol Chem.2004;279:44303-44310.
13 Yu FH,Westenbroek RE.Sodium channel beta4 a new disulfide-linked auxiliary subunit with similarity to beta2.J Neurescience.2003;23:577-585.
14 Xiao ZC,Ragsdale DS,Malhotra JD,et al.Tenascin-R is a functional modulator of sodium channel beta subunits.J Biol Chem.1999;274:26511-26517.
15 Ratcliffe CF,Westenbroek RE,Curtis R,et al.Sodium channel betal and beta3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain.J Cell Biol.2001;154:427-434.
16 Qu Y,Curtis R,Lawson D,etal.Differential modulation of sodium channel gating and persistent sodium currents by the betal,beta2 and beta3 subunits.Mol Cell Neurosci.2001;18:570-580.
17 Kanai K,Hirose S,Qguni H,et al.Effect of localization of missense mutations in SCNA on epilepsy phenotype severity.Neurology.2004;63:329-334.
18 Cossette P,Loukas A,Lafreniere RG,et al.Funcational characterization of the D188V mution in neuronal voltage-gated sodium channel causing generalized epilepsy with febrile seizures plus (GEFS+).Epilepsy Res.2003;53:107-117.
19 Fujiwara T,Sugawara T.Mutation of sodium channel a subunit type 1 (SCNIA)in intractable childhood epilepsies with frequent generalized tonic-clonic seizures.Brain.2003;126:531-546.
20 Vanoye CG,Lossin C,Rhodes TH,et al.Single-channel properties of human Na_vl.l and mechanism of channel dysfunction in SCNIA-associated epilepsy.J Gen Physiol.2006;127:1-14.
21 Morimoto M,Mazaki E,Nishimura A,et al.SCNIA mutation mosaicism in a family with severe myoclonic epilepsy in infancy.Epilepsia.2006;47:1732-1736.
22 Jansen FE,Sadleir LG,Harkin LA,et al.Severe myoclonic epilepsy of infancy (Dravet syndrome):recognition and diagnosis in adults.Neurology.2006;67:2224-2226.
23 Barela AJ,Waddy SP,Lickfett JG et al.An epilepsy mutation in the sodium channel SCNIA that decreases channel excitability.J Neurosci.2006;26:2714-2723.
24 Kearney JA,Plummer NW.A gain of function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities.Neuroscience.2001;102:307-317.
25 Sugawara T,Tsurubuchi Y,Agarwala KL,et al.A missense mutation of the Na~+ channel α_2 subunit gene Na(V)1.2 in a patient with febrile and a febrile seizures causes channel dysfunction.Proc Natl Acad Sci USA.2001;98:6384-6389.
26 Kamiya K,Kaneda M.A nonsense mutation of the sodium channel gene SCN2A in a patient with intractable epilepsy and mental decline.J Neurosci.2004;24:2690-2698.
27 Striano P,Bordo L,Lispi ML,et al.A Novel SCN2A Mutation in Family with Benign Familial Infantile Seizures.Epilepsia.2006;47:218-220.
28 Copley RR.Evolutionary convergence of alternative splicing in ion channels.Trends Genet.2004;20:171-176.
29 Goldin AL.Evolution of voltage-gated Na~+ channels.J Exp Biol.2002;205:575-584.
30 Wallace RH,Wang DW,Singh R,et al.Febrile seizures and generalized epilepsy associated with a mutation in the Na~+-channel β_1 subunit gene SCN1B.Nat Genet.1998;19:366-370.
31 Moran O,Conti F.Skeletal muscle sodium channel is affected by an epileptogenic β_1 subunit mutation.Biochem.2001;282:55-59.
32 Meadows LS,Malhotra J,Loukas A,et al.Functional and Biochemical Analysis of a Sodium Channel β_1 Subunit Mutation Responsible for Generalized Epilepsy with Febrile Seizures Plus Type 1.Neuroscience.2002;22:10699-10709.
33 Gorter JA,Van vliet EA,Lopes da Silva FH,et al.Sodium channel β_1 subunit expression is increased in reactive astrocytes in a rat model for mesial temporal lobe epilepsy.Neuroscience.2002;16:1211-1220.
34 Chen C,Bharucha V,Chen Y,et al.Reduced sodium channel density,altered voltage dependence of inactivation,and increased susceptibility to seizures in mice lacking sodium channel P2-subunits.Proc Natl Acad Sci USA.2002;99:17072-17077.
35 Ellerkmann RK,Remy S,Chen J,et al.Molecular and functional changes in voltage-dependent Na(+)channels following pilocarpine-induced status epilepticus in rat dentate granule cells.Neuroscience.2003;119:323-333.
36 Lucas PT,Meadows LS,Nicholls J,et al.An epilepsy mutation in the betal subunit of the voltage-gated sodium channel results in reduced channel sensitivity to phenytoin.Epilepsy Res. 2005;64(3):77-84.
37 Scheffer IE,Harkin LA,Grinton BE,et al.Temporal lobe epilepsy and GEFS+ phenotypes associated with SCNIB mutations.Brain.2007;130:100-109.
2 Hirose S,Okada M,Kaneko S,et al.Are some idiopathic epilepsies disorders of ion channels? A working hypothesis.Epilepsy Research.2000;41:191-194.
3 Catterall WA.Molecular properties of brain sodium channels:an important target for anticonvulsants.Adv Neurology.1999;79:441-456.
4 Johnson D,Montpetit ML,Stocker PJ,et al.The sialic acid component of the β_1 subunit modulates voltage-gated sodium channel function.J Biol Chem.2004;279:44303-44310.
5 Meisler MH and Kearney JA.Sodium channel mutations in epilepsy and other neurological disorders.J Clin Invest.2005;115:2010-2017.
6 Audenaert D,Claes L,Ceulemans B,et al.A deletion in SCN1B is associated with febrile seizures and early-onset absence epilepsy.Neurology.2003;61:854-856.
7 Lucas PT,Meadows LS,Nicholls J,et al.An epilepsy mutation in the betal subunit of the voltage-gated sodium channel results in reduced channel sensitivity to phenytoin.Epilepsy Res.2005;64:77-84.
8 Scheffer IE,Harkin LA,Grinton BE,et al.Temporal lobe epilepsy and GEFS~+ phenotypes associated with SCN1B mutations.Brain.2007;130:100-109.
9 Kanai K,Hirose S,Qguni H,et al.Effect of localization of missense mutations in SCN1A on epilepsy phenotype severity.Neurology.2004;63:329-334.
10 Cossette P,Loukas A,Lafreniere RG,et al.Funcational characterization of the D188V mution in neuronal voltage-gated sodium channel causing generalized epilepsy with febrile seizures plus (GEFS~+).Epilepsy Res.2003;53:107-117.
11 Vanoye CG,Lossin C,Rhodes TH,et al.Single-channel properties of human Na_vl.l and mechanism of channel dysfunction in SCN1A-associated epilepsy.J Gen Physiol.2006;127:1-14.
12 Fujiwara T,Sugawara T.Mutation of sodium channel a subunit type 1(SCN1A)in intractable childhood epilepsies with frequent generalized tonic-clonic seizures.Brain.2003;126:531-546.
13 Morimoto M,Mazaki E,Nishimura A,et al.SCN1A mutation mosaicism in a family with severe myoclonic epilepsy in infancy.Epilepsia.2006;47:1732-1736.
14 Jansen FE,Sadleir LG,Harkin LA,et al.Severe myoclonic epilepsy of infancy (Dravet syndrome):recognition and diagnosis in adults.Neurology.2006;67:2224-2226.
15 Barela AJ,Waddy SP,Lickfett JG,et al.An epilepsy mutation in the sodium channel SCN1A that decreases channel excitability.J Neurosci.2006;26:2714-2723.
16 Mantegazza M,Gambardella A,Rusconi R,et al.Identification of a Na_v1.1 sodium channel (SCN1A)loss-of-function mutation associated with familial simple febrile seizures.Proc Natl Acad Sci USA.2005;102:18177-18182.
17 Yu FH,Mantegazza M,Westenbroek RE,et al.Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy.Nat Neurosci.2006;9:1142-1149.
18 Serikawa T,Yamada J.Epileptic seizures in rats homozygous for two mutations,zitter and tremor.J Hered.1986;77:441-444.
19 Ji-qun C,Ishihara K,Nagayama T,et al.Long-lasting antiepileptic effects of levetiracetam against epileptic seizures in the spontaneously epileptic rat (SER):differentiation of levetiracetam from conventional antiepileptic drugs.Epilepsia.2005;46:1362-1370.
20 Yan HD,Ji-qun C,Ishihara K,et al.Separation of antiepileptogenic and antiseizure effects of levetiracetam in the spontaneously epileptic rat (SER).Epilepsia.2005;46:1170-1177.
21 Seki T,Matsubayashi H,Amano T,et al.Adenoviral gene transfer of aspartoacylase ameliorates tonic convulsions of spontaneously epileptic rats.Neurochem Int.2004;45:171-178.
22 Amano T,Aihua Z,Matsubayashi H,et al.Antiepileptic effects of single and repeated oral administrations of S-312-d,a novel calcium channel antagonist,on tonic convulsions in spontaneously epileptic rats.J Pharmacol Sci.2004;195:355-362.
23 Amano T,Amano H,Matsubayashi H,et al.Enhanced Ca(2+)influx with mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rats.Brain Res.2001;910:199-203.
24 Akopian AN,Souslova V,England S,et al.The tetrodotoxin-resistent sodium channel SNS has a specialized function in pain pathways.Nature Neuroscience.1999;2:541-548.
25 Chen J,Sochivko D,Beck H,et al.Activity-induced expression of common reference genes in individual ens neurons.Lab Invest.2001;81:913-916.
26 Ellerkmann RK,Remy S,Chen J,etal.Molecular and functional changes in voltage-dependent Na(+)channels following pilocarpine-induced status epilepticus in rat dentate granule cells.Neuroscience.2003;119:323-333.
27 Kalume F,Yu FH,Westenbroek,et al.Reduced sodium current in Purkinje neurons from Nay 1.1 mutant mice:implications for ataxia in severe myoclonic epilepsy in infancy.J Neurosci.2007;27:11065-11074.
28 Klein JP,Khera DS,Nersesyan H,et al.Dysregulation of sodium channel expression in cortical neurons in a rodent model of absence epilepsy,Brain Res.2004;1000:102-109.
29 Whitaker WR,Faull RL,Dragunow M,et al.Changes in the mRNAs encoding voltage-gated sodium channel type Ⅱ and Ⅲ in human epileptic hippocampus,Neuroscience.2001;106:275-285.
30 Aronica E,Yankaya B,Troost D,et al.Induction of neonatal sodium channel Ⅱ and Ⅲ a-isoform mRNAs in neurons and microglia after status epilepticus in the rat hippocampus,Eur J Neurosci.2001;13:1261-1266.
31 Copley RR,Evolutionary convergence of alternative splicing in ion channels.Trends Genet.2004;20:171-176.
32 Felts PA,Yokoyama S,Dib-Hajj S,et al.Sodium channel alpha-subunit mRNAs Ⅰ,Ⅱ,Ⅲ,NaG,Na6 and hNE (PN1):different expression patterns in developing rat nervous system.Mol Brain Res.1997;45:71-82.
33 Goldin AL.Evolution of voltage-gated Na~+ channels.J Exp Biol.2002;205:575-584.
34 Aronica E,Yankaya B,Troost D,et al.Induction of neonatal sodium channel Ⅱ and Ⅲ a-isoform mRNAs in neurons and microglia after status epilepticus in the rat hippocampus.Eur J Neurosci.2001;13:1261-1266.
35 Yu FH,Mantegazza M,Westenbroek RE,et al.Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy.Nat Neurosci.2006;9: 1142-1149.
36 Ito M,Shirasaka Y,Hirose S,et al.Seizure phenotypes of a family with missense mutations in SCN2A.Pediatr Neurol.2004;31:150-152.
37 Striano,Bordo L,Lispi ML,et al.A novel SCN2A mutation in family with benign familial infantile seizures.Epilepsia.2006;47:218-220.
38 Misra SN,Kahlig KM,George AL Jr.Impaired Na_v1.2 function and reduced cell surface expression in benign familial neonatal-infantile seizures.Epilepsia.2008;49:1535-1545.
39 Scalmani P,Rusconi R,Armatura E,et al.Effects in neocortical neurons of mutations of the Na(v)1.2 Na~+ channel causing benign familial neonatal-infantile seizures.J Neurosci.2006;26:10100-10109.
40 Xu R,Thomas EA,Jenkins M,et al.A childhood epilepsy mutation reveals a role for developmentally regulated splicing of a sodium channel.Mol Cell Neurosci.2007;35:292-301.
41 Morgan K,Stevens EB,Shah B,et al.Beta 3:an additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics.Proc Natl Acad Sci USA.2000;97:2308-2313.
42 Johnson D,Montpetit ML,Stocker PJ,et al.The sialic acid component of the β_1 subunit modulates voltage-gated sodium channel function.J Biol Chem.2004;279:44303-44310.
43 Dyke PM,Laurence SM,Chen CL,et al.Sodium Channel β_1 subunit mediated modulation of Na_v1.2 currents and cell surface density is dependent on interactions with contactin and ankyrin.J Biol Chem.2004;279:16044-16049.
44 Malhotra JD,Kazen-Gillespie K,Hortsch M,et al.Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact.J Biol Chem.2000;275:11383-11388.
45 Makita N,Sloan-Brown K,Weghuis DO,et al.Genomic organization and chromosomal assignment of the human voltage-gated Na(+)channel beta-1 subunit gene (SCN1B).Genomics.1994;23:628-634.
46 Isom LL.Sodium channel beta subunits:anything but auxiliary.Neuroscientist.2001;7:42-54.
47 Wallace RH,Wang DW,Singh R,et al.Febrile seizures and generalized epilepsy associated with a mutation in the Na~+ channel β_1 subunit gene SCN1B.Nat Genet.1998;19:366-370.
48 Wallace RH,Scheffer IE,Parasivam G,et al.Generalized epilepsy with febrile seizures plus:mutation of the sodium channel subunit SCN1B.Neurology.2002;58:1426-1429.
49 Scheffer IE,Harkin LA,Grinton BE,et al.Temporal lobe epilepsy and GEFS+ phenotypes associated with SCN1B mutations.Brain.2007;130:100-109.
50 Fjelll J,Dib-Hajj S,Fried K,et al.Differential expression of sodium channel genes in retinal ganglion cells.Brain Res Mol Brain Res.1997;15:197-204.
51 Moran O,Conti F.Skeletal muscle sodium channel is affected by an epileptogenic β_1 subunit mutation.Biochem.2001;282:55-59.
52 Meadows LS,Malhotra J,Loukas A,et al.Functional and biochemical analysis of a sodium channel β_1 subunit mutation responsible for generalized epilepsy with febrile seizures plus type 1.Neuroscience.2002;22:10699-10709.
53 Gorter JA,van Vliet EA,Lopes da Silva FH,et al.Sodium channel p,subunit expression is increased in reactive astrocytes in a rat model for mesial temporal lobe epilepsy.Eur J Neurosci.2002;16:360-364.
54 Chen CL,Ruth E,Xu XR,et al.Mice lacking sodium channel β_1 subunits display defects in neuronal excitability,sodium channel expression and nodal architecture.Neuroscience.2004;24:4030-4042.
1 K(o|¨)hling R.Voltage-gated sodium channels in epilepsy.Epilepsia.2002;43:1278-1295.
2 Hirose S,Okada M,Kaneko S,et al.Are some idiopathic epilepsies disorders of ion channels? A working hypothesis.Epilepsy Research.2000;41:191-194.
3 Catterall WA.Molecular properties of brain sodium channels:an important target for anticonvulsants.Adv Neurology.1999;79:441-456.
4 Johnson D,Montpetit ML,Stocker PJ,et al.The sialic acid component of the β_1 subunit modulates voltage-gated sodium channel function.J Biol Chem.2004;279:44303-44310.
5 Meisler MH and Kearney JA.Sodium channel mutations in epilepsy and other neurological disorders.J Clin Invest.2005;115:2010-2017.
6 Malhotra JD,Kazen-Gillespie K,Hortsch M,et al.Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact.J Biol Chem.2000;275:11383-11388.
7 Makita N,Sloan-Brown K,Weghuis DO,et al.Genomic organization and chromosomal assignment of the human voltage-gated Na(+)channel beta-1 subunit gene (SCN1B).Genomics.1994;23:628-634.
8 Morgan K,Stevens EB,Shah B,et al.Beta 3:an additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics.Proc Natl Acad Sci USA.2000;97:2308-2313.
9 Isom LL.Sodium Channel p Subunits:Anything but Auxiliary.Neuroscientist.2001;7:42-54.
10 Chen CL,Ruth E,Xu XR,et al.Mice Lacking Sodium Channel β_1 Subunits Display Defects in Neuronal Excitability,Sodium Channel Expression,and Nodal Architecture.Neuroscience.2004;24:4030-4042.
11 Meadows LS,Chen YH,Powell AJ,et al.Functional modulation of human brain Navl.3 sodium channels,expressed in mammalian cells,by auxiliary beta 1,beta 2 and beta 3 subunits.Neuroscience.2002;114:745-753.
12 Johnson D,Montpetit ML,Stocker PJ,et al.The Sialic Acid Component of the β_1 Subunit Modulates Voltage-gated Sodium Channel Function.J Biol Chem.2004;279:44303-44310.
13 Yu FH,Westenbroek RE.Sodium channel beta4 a new disulfide-linked auxiliary subunit with similarity to beta2.J Neurescience.2003;23:577-585.
14 Xiao ZC,Ragsdale DS,Malhotra JD,et al.Tenascin-R is a functional modulator of sodium channel beta subunits.J Biol Chem.1999;274:26511-26517.
15 Ratcliffe CF,Westenbroek RE,Curtis R,et al.Sodium channel betal and beta3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain.J Cell Biol.2001;154:427-434.
16 Qu Y,Curtis R,Lawson D,etal.Differential modulation of sodium channel gating and persistent sodium currents by the betal,beta2 and beta3 subunits.Mol Cell Neurosci.2001;18:570-580.
17 Kanai K,Hirose S,Qguni H,et al.Effect of localization of missense mutations in SCNA on epilepsy phenotype severity.Neurology.2004;63:329-334.
18 Cossette P,Loukas A,Lafreniere RG,et al.Funcational characterization of the D188V mution in neuronal voltage-gated sodium channel causing generalized epilepsy with febrile seizures plus (GEFS+).Epilepsy Res.2003;53:107-117.
19 Fujiwara T,Sugawara T.Mutation of sodium channel a subunit type 1 (SCNIA)in intractable childhood epilepsies with frequent generalized tonic-clonic seizures.Brain.2003;126:531-546.
20 Vanoye CG,Lossin C,Rhodes TH,et al.Single-channel properties of human Na_vl.l and mechanism of channel dysfunction in SCNIA-associated epilepsy.J Gen Physiol.2006;127:1-14.
21 Morimoto M,Mazaki E,Nishimura A,et al.SCNIA mutation mosaicism in a family with severe myoclonic epilepsy in infancy.Epilepsia.2006;47:1732-1736.
22 Jansen FE,Sadleir LG,Harkin LA,et al.Severe myoclonic epilepsy of infancy (Dravet syndrome):recognition and diagnosis in adults.Neurology.2006;67:2224-2226.
23 Barela AJ,Waddy SP,Lickfett JG et al.An epilepsy mutation in the sodium channel SCNIA that decreases channel excitability.J Neurosci.2006;26:2714-2723.
24 Kearney JA,Plummer NW.A gain of function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities.Neuroscience.2001;102:307-317.
25 Sugawara T,Tsurubuchi Y,Agarwala KL,et al.A missense mutation of the Na~+ channel α_2 subunit gene Na(V)1.2 in a patient with febrile and a febrile seizures causes channel dysfunction.Proc Natl Acad Sci USA.2001;98:6384-6389.
26 Kamiya K,Kaneda M.A nonsense mutation of the sodium channel gene SCN2A in a patient with intractable epilepsy and mental decline.J Neurosci.2004;24:2690-2698.
27 Striano P,Bordo L,Lispi ML,et al.A Novel SCN2A Mutation in Family with Benign Familial Infantile Seizures.Epilepsia.2006;47:218-220.
28 Copley RR.Evolutionary convergence of alternative splicing in ion channels.Trends Genet.2004;20:171-176.
29 Goldin AL.Evolution of voltage-gated Na~+ channels.J Exp Biol.2002;205:575-584.
30 Wallace RH,Wang DW,Singh R,et al.Febrile seizures and generalized epilepsy associated with a mutation in the Na~+-channel β_1 subunit gene SCN1B.Nat Genet.1998;19:366-370.
31 Moran O,Conti F.Skeletal muscle sodium channel is affected by an epileptogenic β_1 subunit mutation.Biochem.2001;282:55-59.
32 Meadows LS,Malhotra J,Loukas A,et al.Functional and Biochemical Analysis of a Sodium Channel β_1 Subunit Mutation Responsible for Generalized Epilepsy with Febrile Seizures Plus Type 1.Neuroscience.2002;22:10699-10709.
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