1996 Fiscal Year Annual Research Report

ATP感受性Kチャネル開口剤の新しい作用機序の分類と臓器作用検定法の開発

Research Project

Project/Area Number	06557044
Research Institution	Osaka University
Principal Investigator	倉智嘉久大阪大学, 医学部, 教授 (30142011)
Co-Investigator(Kenkyū-buntansha)	山田充彦大阪大学, 医学部, 助手 (10263237) 内匠透大阪大学, 医学部, 助手 (00222092) 堀正二大阪大学, 医学部, 教授 (20124779)
Keywords	ATP感受性カリウムチャネル / 心臓 / 平滑筋 / クローニング / スルフオニル尿素受容体 / ATP / カリウムチャネルオープナ- / 培養細胞
Research Abstract	ATP感受性カリウムチャネル(K_<ATP>)に対するカリウムチャネルオープナ-(KCO)の効果はこのチャネルのサブタイプによって異なっている。この分子機構を解明し、KCOの開発の論理的な基礎を構築するため、脳cDNAライブラリーからサブユニットであるBIRとuK_<ATP>をクローン化した。また、心臓からスルフオニル尿素受容体SUR2A,SUR2BcDNAを新たにクローン化した。SUR2BはdifferentialsplicingによってSUR2Aと同じ遺伝子から作られると考えられ、C末端の42個のアミノ酸配列はSUR1に似ていた。SUR2A,SUR2B共に培養細胞(HEK)においてBIRもしくはuK_<ATP>とともに共発現させることによってK_<ATP>が出現した。SUR2Bの発見は広く分布しており、uK_<ATP>と共に培養細胞HEKに発現させたところ、ATPの阻害効果はMg-ATPcomplexの阻害効果よりも強いことや、ピナシジルやジアゾキサイドが共によく効く等、血管平滑筋に存在するATP感受性カリウムチャネルに非常に似た性質のチャネルを発現することがわかった。これははじめての血管平滑筋型ATP感受性カリウムチャネルのクローニングである。一方、SUR2AとBIRを構成サブユニットとするK_<ATP>は心臓のK_<ATP>チャネルの性質と非常に似ていた。即ち、ATPの阻害効果はMg-ATPcomplexの阻害効果とほぼ同等であり、一方、KCOの効果ではジアゾキサイドに反応しにくく、ピナシジルはよく効くことなど心筋K_<ATP>に非常によく似ていた。SUR2AとBIRによるK_<ATP>はSUR2BとBIRのチャネルに比べニコランジルの効果が約1/10と効きにくいことが判明した。また、BIRとuK_<ATP>のキメラによる解析から2つの膜貫通領域とそれにはさまれた領域がコンダクタンスの大きさを決定することがわかった。以上のように、K_<ATP>のクローンを培養細胞に発現させてパッチクランプ法で測定することによりKCOのK_<ATP>サブタイプ別の効果を客観的に評価する系を確立することができた。

Research Products

(14 results)

All Other

All Publications (14 results)

[Publications] Isomoto S.: "A novel ubiquitously-distributed isoform of GIRK2(GIRK2B) enhances GIRK1 expression of the G-protein-gated K+ current in Xenopus oocytes." Biochemical and Biophysical Research Communications. 218. 286-291 (1996)
[Publications] Horio Y.: "Differential distribution of classical inwardly rectifying potassium channel mRNAs in the brain; comparison of IRK2 with IRK1 and IRK3." FEBS Letters. 379. 239-243 (1996)
[Publications] Morishige K.: "G Protein-gated K+ channel (GIRK1) protein is expressed presynaptically in the paraventricular nucleus of the hypothalamus." Biochemical and Biophysical Research Communications. 220. 300-305 (1996)
[Publications] Terzic A.: "Actin microfilament-distrupters enhance KATP channel opening in patches from guinea-pig cardiomyocytes." Journal of Physiology. 492.2. 395-404 (1996)
[Publications] Ito M.: "Immunolocalization of an inwardly rectifying K+ channel,KAB-2(Kir4.), in the basolateral membrane of renal distal tubular epithelia." FEBS Letters. 388. 11-15 (1996)
[Publications] Yamashita T.: "Competition between Mg2+ and spermine for a cloned IRK2 channel expressed in a human cell line." Journal of Physiology. 493. 143-156 (1996)
[Publications] Isomoto S.: "A novel sulfonylurea receptor forms with BIR(Kir6.2)a smooth muscle type ATP-sensitive K+ channel." The Journal of Biological Chemistry. 271. 24321-24324 (1996)
[Publications] Kurachi Y.: "Molecular and functional heterogeneity of inward rectifier potassium channels in brain and heart." Journal of Cardiac Failure. 2(4S). S59-S62 (1996)
[Publications] Takumi T.: "Assignment of the murine inward rectifier potassium channel Irk2(Kir2.2) gene to the central region of mouse chromosome 11." Genomics. 37. 270-272 (1996)
[Publications] Kondo C.: "Cloning and functional expression of a novel isoform of ROMK inwardly rectifying ATP-dependent K+ channel,ROMK6(Kirl.lf)." FEBS Letters. 399. 122-126 (1996)
[Publications] Hosoya Y.: "A functional model for G protein activation of the muscarinic K+ channel in guinea pig atrial myocytes." Journal of General Physiology. 108. 485-495 (1966)
[Publications] Hazama H.: "Neurokinin a and Ca2+ current induce Ca2+ -activated Cl- currents in guinea-pig tracheal myocytes." Journal of Physiology. 492(2). 377-393 (1996)
[Publications] Nakajima T.: "Endothelin-1 and vasopressin activate Ca2+ -permeable non-selective cation channels in aortic smooth muscle cells: mechanism of receptor-mediated Ca2+ influx." Journal of Molecular and Cellular Cardiology. 28. 707-722 (1966)
[Publications] Horio Y: "Clustering and enhanced activity of an inwardly rectifying potassium channel,Kir4.1,by an anchoring protein,PSD-95/SAP90" The Journal of Biologycal Chemistry. (in press).

1996 Fiscal Year Annual Research Report

ATP感受性Kチャネル開口剤の新しい作用機序の分類と臓器作用検定法の開発

Principal Investigator

倉智 嘉久 大阪大学, 医学部, 教授 (30142011)

Research Products

[Publications] Isomoto S.: "A novel ubiquitously-distributed isoform of GIRK2(GIRK2B) enhances GIRK1 expression of the G-protein-gated K+ current in Xenopus oocytes." Biochemical and Biophysical Research Communications. 218. 286-291 (1996)

[Publications] Horio Y.: "Differential distribution of classical inwardly rectifying potassium channel mRNAs in the brain; comparison of IRK2 with IRK1 and IRK3." FEBS Letters. 379. 239-243 (1996)

[Publications] Morishige K.: "G Protein-gated K+ channel (GIRK1) protein is expressed presynaptically in the paraventricular nucleus of the hypothalamus." Biochemical and Biophysical Research Communications. 220. 300-305 (1996)

[Publications] Terzic A.: "Actin microfilament-distrupters enhance KATP channel opening in patches from guinea-pig cardiomyocytes." Journal of Physiology. 492.2. 395-404 (1996)

[Publications] Ito M.: "Immunolocalization of an inwardly rectifying K+ channel,KAB-2(Kir4.), in the basolateral membrane of renal distal tubular epithelia." FEBS Letters. 388. 11-15 (1996)

[Publications] Yamashita T.: "Competition between Mg2+ and spermine for a cloned IRK2 channel expressed in a human cell line." Journal of Physiology. 493. 143-156 (1996)

[Publications] Isomoto S.: "A novel sulfonylurea receptor forms with BIR(Kir6.2)a smooth muscle type ATP-sensitive K+ channel." The Journal of Biological Chemistry. 271. 24321-24324 (1996)

[Publications] Kurachi Y.: "Molecular and functional heterogeneity of inward rectifier potassium channels in brain and heart." Journal of Cardiac Failure. 2(4S). S59-S62 (1996)

[Publications] Takumi T.: "Assignment of the murine inward rectifier potassium channel Irk2(Kir2.2) gene to the central region of mouse chromosome 11." Genomics. 37. 270-272 (1996)

[Publications] Kondo C.: "Cloning and functional expression of a novel isoform of ROMK inwardly rectifying ATP-dependent K+ channel,ROMK6(Kirl.lf)." FEBS Letters. 399. 122-126 (1996)

[Publications] Hosoya Y.: "A functional model for G protein activation of the muscarinic K+ channel in guinea pig atrial myocytes." Journal of General Physiology. 108. 485-495 (1966)

[Publications] Hazama H.: "Neurokinin a and Ca2+ current induce Ca2+ -activated Cl- currents in guinea-pig tracheal myocytes." Journal of Physiology. 492(2). 377-393 (1996)

[Publications] Nakajima T.: "Endothelin-1 and vasopressin activate Ca2+ -permeable non-selective cation channels in aortic smooth muscle cells: mechanism of receptor-mediated Ca2+ influx." Journal of Molecular and Cellular Cardiology. 28. 707-722 (1966)

[Publications] Horio Y: "Clustering and enhanced activity of an inwardly rectifying potassium channel,Kir4.1,by an anchoring protein,PSD-95/SAP90" The Journal of Biologycal Chemistry. (in press).

倉智嘉久大阪大学, 医学部, 教授 (30142011)