Molecular Basis of Sodium Channel Assembly in Neural Cells

神经细胞钠通道组装的分子基础

• 批准号: 07044223
• 负责人: TAKAHASHI Kunitaro
• 金额: $ 3.9万
• 依托单位: Meiji College of Pharmacy
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044223/
• 关键词: Ascidian Embryo; Na Channel Regulation; TuNaI; TTX-sensitive Mutation; GJC Expression; Cell-cell-Interaction; Two-Hybrid; GFP-fused Protein

When voltage-dependent Na channel, one of specific characteristics of neuronal cells, is functionally expressed in newly developed neurons, it is necessary that the channel proteins are properly and timely assembled in the membranes at various locations of the cell body. In the present project we have aimed to elucidate the molecular regulatory mechanisms of assembly of Na channels by using a specially designed preparation of an interacting and neurally inducing two-embryonic-cell-system separated from the early ascidian embryo which was reported previously by us. In order to analyze the regulation at gene expression level, a putative Na+channel cDNA,TuNal, was cloned from embryos of an ascidian, H.roretzi (Okamura et al.1994). For the purpose of distinguishing the sodium current encoded by exogenously introduced TuNal from endogeneous TTX-resistant one, a TTX-sensitive point mutation was introduced. And we overexpressed the mutated TuNal in neuronally differentiated blastomere by inje … More cting the encoding mRNA.This blastomere showed almost no significant increase in total Na+current although TTX-resistant component was clearly pbserved. Interestingly. K+current was increased in the injected cells, indicating that K+ channel expression is regulated coordinately with Na+ channel expression. Although previously reported that gap junctional communication (GJC) between the interacting and neuron-inducing cell pair increased until 20-30 developmental hr and then suddenly disappeared, treatment with a kinase inhibitor (K252a) delayd the disappearance of GJC for up to 80 hr ; followed by a similar delay in expression of neuronal characteristics, such as Na and K channels. Furthermore when connexin 32 cDNA was injected into the neuronally determined cell and the expression of GJC was forced, neuronal expression was suppressed. Recently, in order to locate the functional GJP channels we are trying to inject synthesized mRNA encoding the fused protein between GJP and green fluorescent proteins. All molecular biological techniques used in the present experiment wre instructed by the collaborative researcher, Professor Gail Mandel in USA. Less

电压依赖性钠通道是神经细胞的特征之一，在新生发育的神经元中要实现功能表达，通道蛋白必须在细胞体各部位的膜上正确、及时地组装。在本项目中，我们的目的是通过从我们之前报道的早期海鞘胚胎中分离出一个特殊设计的相互作用和神经诱导的双胚胎细胞系统来阐明Na通道组装的分子调控机制。为了分析基因表达水平上的调控，从海鞘H.roretzi胚胎中克隆了一个推测的Na+通道cDNA TuNal （Okamura et al.1994）。为了区分外源导入的TuNal编码的钠电流与内源ttx抗性的钠电流，引入了ttx敏感点突变。我们通过注射编码mRNA，在神经分化的卵裂球中过表达突变的TuNal。尽管有明显的抗ttx成分，但卵裂球的总Na+电流几乎没有显著增加。有趣的是。注射细胞中K+电流增加，表明K+通道的表达与Na+通道的表达是协调调节的。虽然先前报道相互作用细胞对和神经元诱导细胞对之间的间隙连接通讯（GJC）增加到20-30发育小时，然后突然消失，但用激酶抑制剂（K252a）治疗将GJC的消失延迟了80小时；随后是神经元特征（如Na和K通道）表达的类似延迟。此外，将连接蛋白32 cDNA注射到神经元确定的细胞中，强迫GJC的表达，抑制神经元的表达。最近，为了定位功能性GJP通道，我们尝试注入合成的编码GJP与绿色荧光蛋白融合蛋白的mRNA。本实验中使用的所有分子生物学技术均由合作研究人员美国Gail Mandel教授指导。少

项目成果

Okada,T.& Okamura,Y.: "Identification of neuronal cells by detecting ion channel mRNA in the larva of the ascidian,Halocynthia roretzi." Zoological Science. 13 suppl. 95 (1996)

冈田，T.

Saitoe,M.,Inazawa,T.& TAKAHASHI,K.: "Neuronal expression in cleavage-arrested ascidian blastomeres requires gap junctional uncoupling from neighbouring cells." Journal of Physiology (Lond.). 491. 825-842 (1996)

Saitoe，M.，稻泽，T.

Okamura, Y., Ono, F.& Okagaki, R.: Japan Scientific Societies Press. Regulation of sodium channel gene expression by cell-specific interactions in the ascidian embryo. In Basic Neuroscience in invertebrates, ed by Koike, H.et al., 21-30 (1996)

冈村，Y.，小野，F.

Funabiki K., Mishina M.& Hirano T: "Retarded vestibular compensation in mutant mice deficient in delta 2 glutamate receptor subunit." Neuroreport. 7 (1). 189-192 (1995)

船引 K.，三品 M.

Okamura,Y.,Ono,F.& Okagaki,R.: "Regulation of sodium channel gene expression by celI-specific interactions in the ascidian embryo.In Basic Neuroscience in lnvertebrates,ed by Koike,H.et al." Japan Scientific Societies Press, 367 (1996)

冈村，Y.，小野，F.