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

使用 RT-PCR 克隆了几种平滑肌组织中负责在去极化时缓慢激活外向电流（IsK 或 mini K）的蛋白质，并使用 RNase 保护测定法检查了其分布。RT-PCR 方法的技术由英国大学教授。编码IsK蛋白的cDNA克隆于大鼠主动脉、十二指肠、虹膜、回肠、胃、气管和膀胱以及兔主动脉、克隆、胃和膀胱膀胱与大鼠肾脏中报道的结果 100% 相同。RNA 保护测定表明，在注射 IsK cRNA 的非洲爪蟾卵母细胞去极化后，IsK 的表达程度相似。兔肠系膜血管床的灌注准备表明，Ba^2+敏感的内向整流K型电流是静息膜电位和静息膜电位的部分原因。在分离的肠系膜动脉肌细胞中，在全细胞电压钳条件下观察到对Ba^<2+>敏感的小K电流（IRK1）。采用RT-PCR方法。以兔心脏的IRK1 cDNA序列为模板，以提取的总RNA为模板，设计引物。从有内皮或无内皮的动脉中获得 RBMAIK1，通过限制性核酸内切酶将 RBMAIK1 分成三个片段，然后将所有片段亚克隆到质粒载体中，然后对所有片段进行测序。将来自 RBMAIK1 cDNA 的 cRNA 注射到非洲爪蟾卵母细胞中导致 Ba^<2+> 的表达敏感的内向整流K电流。哺乳动物细胞系的基因转染技术将从卡尔加里大学转移到Watanabe的小组。已经报道了包括门静脉在内的几种类型的平滑肌细胞中存在A型K电流。 Kv4.2 或 Kv1.4 是大鼠心脏中负责 A 型 K 电流的主要 K 通道。为了鉴定负责平滑肌中的 K 通道，使用几种类型的总 RNA 进行了 RT-PCR。以大鼠脑部的Kv4.2和1.4为模板，设计引物，得到约1.7Kbps的PCR产物，并对PCR产物进行亚克隆和测序。