Molecular biology of vascular and cordiac k channels

用RT-PCR方法克隆了几种平滑肌组织中负责去极化时缓慢激活外向电流的蛋白(ISK或mini K)，并用核糖核酸酶保护实验检测了其分布。RT-PCR技术在卡尔加里大学教授。在大鼠主动脉、十二指肠、虹膜、回肠、胃、气管和膀胱以及兔的主动脉、克隆体、胃和膀胱中克隆的ISK蛋白编码基因与已报道的大鼠肾脏中的同源性为100%。RNA保护实验表明，ISK在所有组织中的表达程度相似。用兔肠系膜血管床灌流制剂进行的药理实验表明，Ba2+敏感的内向整流型K电流对肠系膜动脉平滑肌的静息膜电位和张力有部分作用。分离的肠系膜动脉肌细胞…此外，在全细胞电压钳下观察到对Ba2+敏感的小K电流。用RT-PCR技术从兔肠系膜动脉中克隆了内向整流型K通道基因(IRK1)。根据兔心脏IRK1基因的序列设计了相应的引物。扩增产物为1392bps(RBMAIK1)，分别来自有或无内皮动脉。用限制性内切酶将RBMAIK1分成3个片段，亚克隆到载体上。然后，所有的片段都被测序了。RBMAIK1与兔心IRK1的同源性为100%。将RBMAIK1基因的cRNA注入非洲爪哇卵母细胞后，表达了敏感的内向整流钾电流。将基因导入哺乳动物细胞系的技术将从卡尔加里大学的渡边组转移到渡边组。已有报道在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A型K电流的主要钾通道。以大鼠脑组织Kv4.2和Kv1.4中设计的引物为模板，以几种类型的平滑肌组织总RNA为模板，进行RT-PCR，以确定在大鼠脑组织中与钾通道有关的钾通道。获得了约1.7Kbps的扩增产物。将对扩增产物进行亚克隆和测序。较少

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