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

(1)我们首先对大鼠主动脉、肠系膜动脉、脑动脉和大脑中已知的VDCC亚型，尤其是T型亚型，以及α1G亚型的转录本进行了RT-PCR检测。 (2)然后，我们从大鼠肠系膜动脉周围区域切下的约3000个片段构建了cDNA文库，通过使用该文库作为模板，通过PCR扩增证实了α1G和α1H的存在。由此扩增 α1H 同工型的剪接变体为此，将野生型 α1H 的全长分为 6 个具有一些碱基重叠的区域，为每个区域设计了特定的引物对，并使用这些引物和对应的 DNA 片段进行扩增。获得了除 N 末端（包括转录起始位点）和 C 末端之外的四个中间区域。对这些 DNA 片段的直接测序揭示了其中的几个重要突变（4）我们现在正在对这四个区域进行序列比较。 DNA 片段和最近从人子宫平滑肌克隆的 α1H 剪接变体，以发现任何显着的相似性，与此同时，现在正在彻底研究 HEK293 细胞中表达的每个人 α1H 剪接变体的电生理特性，特别是在阈值电位方面。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 连接区中具有缺陷的变体）显示。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，因为这将是激活阈值通过其磷酸化原位动态调节的机制。说明并或许可以解释 NICC 的独特门控特性。