Molecular biology of vascular and cordiac k channels

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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