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cDNA cloning of the new nigedipine-insenstive voltage-dependent Ca^2+ channels in the peripheral resistant artery..

外周阻力动脉中新型尼格地平不敏感电压依赖性 Ca^2 通道的 cDNA 克隆。

基本信息

批准号：
14370033
负责人：
ITO Yushi
金额：
$ 8.96万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

We attempted the cDNA cloning of nifedipine-insensitive voltage-dependent Ca^<2+> channels (VDCC) which predominantly distribute in the peripheral mesenteric arteries and exhibit unique properties distinct from those of hitherto-known VDCCs. (1)We first performed a RT-PCR detection of so far known VDCC isoforms, especially of T -type, in rat aorta, mesenteric artery, cerebral artery and brain. While the transcripts of α1G isoform were almost equally amplified from all tissues examined, those of α1H were variable depending on the region of arteries, and all was entirely undetectable. (2)We then constructed a cDNA library from about 3000 segments dissected from the peripheral regions of rat mesenteric artery. By using this library as a template, the presence of both α1G and α1H was confirmed by PCR amplification. (3)We next attempted to amplify the splice variants of α1H isoform from this library. For this purpose, the full length of the wild-type α1H was divided into 6 regions with some … More base overlaps, for each of which specific primer pairs were designed. PCR amplification was performed with these primers, and DNA fragments corresponding to four middle regions excluding the N-terminal (which includes the transcription initiation site)' and C-terminal ends were obtained. Direct sequencing of these DNA fragments revealed several important mutations therein. (4)We are now performing a sequence comparison between the four DNA fragments and the splice variants of α1H isoform recently cloned from human uterine smooth muscle, to find any significant similarities. In parallel with this, the electrophysiological properties of each human α1H splice variant expressed in HEK293 cells are now being thoroughly investigated, especially with respect to the threshold potential for activation and inactivation. We have already found, in collaboration with others, that some α1H splice variants (those having defects in the III-IV linker region) evaluated in the Xenopus oocyte system show appreciable depolarization shifts of activation potential. We will also focus on a possible modulatory effect of calmodulin-dependent kinase II -mediated phosphorylation on α1H channel gating, since this would be a mechanism by which the activation threshold is dynamically regulated in in situ by its phosphorylated state and may perhaps explain the unique gating properties of NICCs. Less

我们试图克隆硝苯地平不敏感的电压依赖性Ca^2+通道（VDCC）的cDNA，该通道主要分布于肠系膜外周动脉，具有不同于已知VDCC的独特性质。(1)We本研究首先对大鼠主动脉、肠系膜动脉、脑动脉和脑中已知的VDCC亚型，特别是T型进行了RT-PCR检测。虽然α1G亚型的转录本在所有检查的组织中几乎同样扩增，但α1H的转录本因动脉区域而异，并且完全检测不到。(2)We然后从大鼠肠系膜动脉外周区切取约3000个片段，构建cDNA文库。以该文库为模板，通过PCR扩增证实了α1G和α1H的存在。(3)We接下来试图从该文库扩增α1H同种型的剪接变体。为此目的，将野生型α1H的全长分为6个区域，其中一些区域是 ...更多信息碱基重叠，为每一个设计特异性引物对。用这些引物进行PCR扩增，获得对应于除N-末端（其包括转录起始位点）和C-末端以外的四个中间区域的DNA片段。这些DNA片段的直接测序揭示了其中的几个重要突变。(4)We目前正在对这四个DNA片段和最近从人类子宫平滑肌中克隆的α1H异构体的剪接变体进行序列比较，以发现任何显著的相似性。与此同时，HEK 293细胞中表达的每个人α1H剪接变体的电生理学特性正在进行彻底研究，特别是关于激活和失活的阈值电位。我们已经与其他人合作发现，在非洲爪蟾卵母细胞系统中评估的一些α1H剪接变体（在III-IV连接区有缺陷的变体）显示出激活电位的明显去极化位移。我们还将关注钙调蛋白依赖性激酶II介导的磷酸化对α1H通道门控的可能调节作用，因为这可能是一种通过磷酸化状态原位动态调节激活阈值的机制，并可能解释NICCs独特的门控特性。少