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Molecular physiological characterization of Ca2+ signal-regulating ion channels that control vectorial substance transport

控制载体物质转运的 Ca2 信号调节离子通道的分子生理特征

基本信息

批准号：
12144210
负责人：
MORI Eyasuo
金额：
$ 22.02万
依托单位：
Kyoto University, Graduate School of Engineering (2003-2004)Okazaki National Research Institutes (2000-2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2004
项目状态：
已结题

项目摘要

We have studied molecular identity of plasma membrane Ca^<2+>-permeable channels and endoplasmic reticulum (ER) Ca^<2+> release channels, and their functional properties, cellular localization, and coupling in epithelial cells, to establish molecular physiological basis underlying vectorization of Ca^<2+> signals in epithelial transport.Abundant expression of receptor-activated TRPC channels was revealed in various epithelial cells. TRPC3, C5, C6, C7 were activated upon stimulation of G-protein-coupled receptors independently of depletion of Ca^<2+> stores/ Ca^<2+> release. TRPC5 was associated with eNOS in the plasma membrane invagination caveola in endothelial cells, and was activated by NO to mediate Ca^<2+> influx that controls a positive feedback loop for receptor-induced NO production cascade. The NAD-activated TRPM2 Ca^<2+> channel was also demonstrated to be linked to cell death under oxidative stress by reactive oxygen/nitrogen species or by TNF.To understand molecular mechani … More sms that determine spatio-temporal patterns of Ca^<2+> signaling, we used chicken DT40 B lymphocytes to create a cell line deficient of TRPC1. The cells showed reduction in store-operated Ca^<2+> (SOC) entry, IP3 ; receptor-mediated Ca^<2+> release, and Ca^<2+> oscillation upon B cell receptor stimulation, suggesting that TRPC1 is critical for patterning Ca^<2+> signals. Furthermore, we demonstrated a positive feedback loop for Ca^<2+>/ IP_3 signaling : translocation and subsequent activation of phospholipase C (PLC)γ elicited by TRPC3-mediating Ca^<2+> entry.For functional coupling between plasmamembrane and ER membrane in Ca^<2+> signaling, the junction structure formed by the two kinds of membrane is essential. To understand molecular architecture of the junction structure, we characterized molecules essential for the structure. Three Junctophilin members have been identified which contribute to formation of the junction structure in excitable cells. A junctional protein Mitsugumin29 plays an important role in ER Ca^<2+> release and SOC. Sarcalmenin in the lumen of sarcoplasmic reticulum (SR) contributes to Ca^<2+> storing capacity of SR. Less

我们研究了质膜Ca^2+通道和内质网Ca^2+释放通道的分子特性、功能特性、细胞定位和偶联，为上皮细胞Ca^2+信号载体化提供分子生理学基础。TRPC 3、C5、C6、C7在刺激G蛋白偶联受体时被激活，而与Ca^<2+>储存/ Ca^<2+>释放的消耗无关。TRPC 5与内皮细胞质膜内陷小窝中的eNOS结合，并被NO激活介导Ca^2+内流，控制受体诱导的NO产生级联反应的正反馈回路。NAD激活的TRPM 2 Ca^<2+>通道也被证明与活性氧/氮或TNF引起的氧化应激下的细胞死亡有关。 ...更多信息为了确定Ca^2+信号传导时空模式，我们使用鸡DT 40 B淋巴细胞建立TRPC 1缺陷细胞系。细胞显示钙池操纵的Ca^2+（SOC）进入、IP 3、受体介导的Ca^2+释放和B细胞受体刺激后的Ca^2+振荡减少，这表明TRPC 1对Ca^2+信号的模式化至关重要。此外，我们还证实了Ca^2+/ IP_3信号通路中存在一个正反馈环：TRPC 3介导的Ca^2+内流导致磷脂酶C（PLC）γ移位并激活，而质膜和内质网膜之间的连接结构是Ca^2+信号通路中质膜和内质网膜功能耦合的关键。为了理解结结构的分子结构，我们表征了结构所必需的分子。已经鉴定了三个亲连接蛋白成员，其有助于可兴奋细胞中连接结构的形成。Mitsugumin 29是一种连接蛋白，在内质网Ca^2+释放和SOC中起重要作用，肌浆网腔中的Sarcalmenin参与了肌浆网的Ca^2+储存。