权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Functions of the C terminal domain of inwardly rectifying K+ channel

内向整流K通道C端域的功能

基本信息

批准号：
11670053
负责人：
OMORI Koichiro
金额：
$ 2.3万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670053/
关键词：
inwardly rectifying K+channel PC12 cell cultured hippocampal neuron adenovirus vector 海馬神経細胞

项目摘要

The inwardly rectifying K+ channels have been found in a variety of cell types including cardiac myocytes and neuronal cells, and play diverse cellular functions such as maintenance of the resting conductance, pacemaker activity, synaptic inhibition, and neuronal firing rates. IRK1, one of the classical inwardly retifying K+ channel, has long C terminal domain comprised of 〜250 amino acidsand the function of the domain has not been elucidated.To analyze the function, we transfected PC12 cells with IRK1 and GFP genes and treated with NGF to differentiate to neuronal cells. Cultured hippocampal neurons from rat fetus were also transfected with the same genes. The gene products could be detected by Western blotting in the PC12 cells but not in the clutured hippocampal neurons. We examined the localization and distribution of the IRK1 gene products by immunohistochemistry in the transfected PC12 cells. The immunoreactivity appeared to be localized mainly in plasma membrane and to a lower extent, in other organelles, which could not been confirmed clearly because of the low expression levels. Then, we used adenovirus vector to introduce IRK1 gene to the cells. IRK1 gene was inserted into the adenovirus vector and the created recombinant adenovirus DNA was used to transfect HEK293 cells to make the recombinant virus particles. The titer of the recombinant virus particles were 〜5x105 pfu/mL.. We used the virus particles to infect PC12 cells and cultured hippocampal neurons at various MOI, but could not get high expression levels owing to low virus titer and high toxicity of the virus solution. We tried to purify the virus particles to remove the toxicity and to get virus with high titer. However, the virus titer was decreased quickly and inactivated during the purification. We suppose the inserted IRK1 gene itself may inhibit the recombinant virus activity.

在包括心肌细胞和神经元细胞在内的多种细胞类型中都发现了向内整流的K+通道，并发挥着多种细胞功能，如维持静息电导、起搏器活性、突触抑制和神经元放电率。IRK1是经典的内修正K+通道之一，具有约250个氨基酸组成的长C末端结构域，其功能尚不清楚。为了分析其功能，我们用IRK1和GFP基因转染PC12细胞，并用NGF处理使其向神经元细胞分化。培养的大鼠胎儿海马神经元也转染了相同的基因。基因产物在PC12细胞中可检测到，但在培养的海马神经元中未检测到。我们通过免疫组化检测了IRK1基因产物在转染PC12细胞中的定位和分布。免疫反应性似乎主要局限于质膜，其他细胞器也有较低程度的反应性，但由于表达水平较低，尚不能明确证实。然后，我们利用腺病毒载体将IRK1基因导入细胞。将IRK1基因插入腺病毒载体，用重组腺病毒DNA转染HEK293细胞制备重组病毒颗粒。重组病毒颗粒滴度为~ 5x105 pfu/mL。我们用病毒颗粒感染PC12细胞，并在不同MOI下培养海马神经元，但由于病毒滴度低，病毒溶液毒性大，无法获得高表达水平。我们试图对病毒颗粒进行纯化，去除毒性，得到高滴度的病毒。但在纯化过程中，病毒滴度迅速下降并灭活。我们推测插入的IRK1基因本身可能抑制重组病毒的活性。