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Molecular biology of vascular and cordiac k channels

血管和心脏 k 通道的分子生物学

基本信息

批准号：
06044192
负责人：
WATANABE Minoru
金额：
$ 4.1万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06044192/
关键词：
IsK RT-PCR TRKI smooth muscle Xenopus oocyte heart A type K current Kv4.2 Kv1.4 抗不整脈薬 PCR法

项目摘要

The protein responsible for slowly activating outward current upon depolarization (IsK or mini K) in several smooth muscle tissues were cloned using RT-PCR and its distribution was examined using RNase protection assay. The techniques of RT-PCR method were taught in The University of Calgary. The cDNA encoding IsK protein cloned in rat aorta, duodenum, iris, ileum, stomach, trachea and urinary bladder and rabbit aorta, clon, stomach and urinary bladder was 100% identical to that has been reported in the rat kidney. The RNA protection assay showed that IsK is expressed in a similar extent in all these tissues. IsK was recorded upon depolarization in Xenopus oocytes injected with IsK cRNA.Pharmacological experiments using perfusion preparations of the rabbit mesenteric vascular bed suggested that Ba^<2+>-sensitive inward rectifier type K current is partly responsible for the resting membrane potential and tone of mesenteric arterial smooth muscle. In isolated mesenteric arterial myocytes … More , small K current which is sensitive to Ba^<2+> was observed under whole-cell voltage clamp. Inward rectifier type K channel (IRK1) was cloned from the rabbit mesenteric artery using RT-PCR.Total RNA extracted from mesenteric artery was used as a template. The primers were designed based on the sequence of IRK1 cDNA of the rabbit heart. A PCR product of 1392 bps (RBMAIK1) was obtained from arteries both with or without endothelium. RBMAIK1 was divided into three fragments by restriction endonucleases and subcloned into a plasmid vector. All fragments were, then, sequenced. RBMAIK1 showed 100% identity with rabbit heart IRK1. The injection of cRNA from the RBMAIK1 cDNA into Xenopus oocytes resulted in expression of Ba^<2+> sensitive inward rectifier K current. The techniques of gene transfection to mammalian cell lines will be transferred to Watanabe's group from that in the University of Calgary.Existence of A-type K currents has been reported in several types of smooth muscle cells including portal vein. Kv4.2 or Kv1.4 is the major K channel responsible for A-type K current in the rat heart. To identify the K channels responsible for those in smooth muscle, RT-PCR was performed using the total RNA of several types of smooth muscle tissues as templates and the primers designed from Kv4.2 and 1.4 of the rat brain. The PCR products of about 1.7Kbps were obtained. The subcloning of the PCR products and sequencing will be carried out. Less

利用RT-PCR技术克隆了几种平滑肌组织中负责去极化后缓慢激活外向电流的蛋白（IsK或miniK），并利用RNase保护试验检测了其分布。RT-PCR技术在加拿大卡尔加里大学教授。在大鼠主动脉、十二指肠、虹膜、回肠、胃、气管、膀胱和兔主动脉、克隆、胃、膀胱中克隆的IsK蛋白cDNA与已报道的大鼠肾脏中IsK蛋白cDNA的同源性为100%。RNA保护试验表明，IsK在所有这些组织中以相似的程度表达。在注射IsK cRNA的非洲爪蟾卵母细胞中记录到IsK去极化后的信号。用兔肠系膜血管床灌注标本进行的药理学实验表明，Ba^<2+>敏感的内向整流型K电流是肠系膜动脉平滑肌静息膜电位和张力的部分原因。在离体肠系膜动脉肌细胞中 ...更多信息在全细胞电压钳下观察到对Ba^<2+>敏感的小K电流。以兔肠系膜动脉总RNA为模板，采用RT-PCR方法从肠系膜动脉中克隆了内向整流型钾通道（IRK 1）基因。根据兔心脏IRK 1 cDNA序列设计引物。从有或无内皮的动脉中获得1392 bp的PCR产物（RBMAIK 1）。RBMAIK 1经限制性内切酶酶切分为3个片段，亚克隆到质粒载体中。然后对所有片段进行测序。RBMAIK 1与兔心脏IRK 1具有100%的同源性。将来自RBMAIK 1 cDNA的cRNA注射到爪蟾卵母细胞中导致Ba^2+敏感性内向整流钾电流的表达。将基因转染到哺乳动物细胞系的技术将从卡尔加里大学的技术转移到Watanabe的小组。A型K电流已在包括门静脉在内的几种平滑肌细胞中报道。Kv4.2或Kv1.4是大鼠心脏中负责A型钾电流的主要钾通道。为了鉴定与平滑肌中的钾通道有关的钾通道，使用几种类型的平滑肌组织的总RNA作为模板，并从大鼠脑的Kv4.2和1.4设计引物，进行RT-PCR。获得了约1.7Kbps的PCR产物。将进行PCR产物的亚克隆和测序。少