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Elucidation of physiological significance of direct Ca^<2+> channel-phospholipase coupling in cell fate control

阐明直接Ca^2通道-磷脂酶偶联在细胞命运控制中的生理意义

基本信息

批准号：
16390076
负责人：
MORI Yasuo
金额：
$ 9.6万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390076/
关键词：
Cell fate Calcium channel Cellular signal receptor calcium signal

项目摘要

TRP superfamily proteins form plasma membrane cation channels acting as ‘sensors' for diverse cellular stimuli. TRPM2 is a Ca^<2+> permeable channel activated by redox statue changes such as oxidative stress. Although it has been suggested that Ca^<2+> influx via TRPM2 mediate cell death, its physiological significance is still elusive. TRPM2 is expressed in immunocytes such as monocytes and lymphocytes. In monocytic cell line U937, it is known that hydrogen peroxide (H_2O_2) induces chemotactic cytokine interleukin-8 (IL-8) production via Erk/NF-κB pathway and that IL-8 is produced via intracellular Ca^<2+> increases. Here, we reveal that oxidative stress-sensitive TRPM2 Ca^<2+> channels are crucial for H_2O_2-induced IL-8 production in U937. We also report physiological significance of the TRPM2 in vivo with regard to the above biological response by utilizing TRPM2 knockout mice.We also demonstrate a novel activation mechanism mediated by cysteine S-nitrosylation in TRP channels. TRPC1, C4, C5, V1, V3, and V4 of TRPC and TRPV families, commonly classified as receptor-activated channels and thermosensor channels, show prominent responses to nitric oxide (NO) to elicit Ca^<2+> entry in HEK293 cells. TRPC5 cysteine mutants, examined for NO responsiveness and susceptibility to S-nitrosylation, have revealed Cys553 and Cys558 as modification sites essential for the NO sensitivity. The responsive TRPs harbor conserved cysteines on the same N-terminal side of the pore region. Strikingly, membrane sidedness of reactive disulfide-induced activation via Cys553 modification suggests cytoplasmic accessibility to Cys553. Thus, multidisciplinary approach based upon chemical biology and physiology identifies the structural motif for NO-sensitive activation gate in TRP channels, establishing ‘NO sensors' as a new functional category extending over different TRP subfamilies.

TRP超家族蛋白形成质膜阳离子通道，作为各种细胞刺激的“传感器”。TRPM2是由氧化应激等氧化还原状态变化激活的Ca^<2+>可渗透通道。虽然有人认为Ca^<2+>通过TRPM2内流介导细胞死亡，但其生理意义尚不明确。TRPM2在免疫细胞如单核细胞和淋巴细胞中表达。在单核细胞系U937中，过氧化氢（H_2O_2）通过Erk/NF-κB途径诱导趋化细胞因子白细胞介素-8 （IL-8）的产生，并通过细胞内Ca^<2+>的增加诱导IL-8的产生。在这里，我们发现氧化应激敏感的TRPM2 Ca^<2+>通道对于h_2o_2诱导的U937中IL-8的产生至关重要。我们还利用TRPM2敲除小鼠报道了TRPM2在体内对上述生物反应的生理意义。我们还证明了一种由半胱氨酸s -亚硝基化介导的TRP通道的新激活机制。TRPC和TRPV家族的TRPC1、C4、C5、V1、V3和V4通常被归类为受体激活通道和热敏通道，它们在HEK293细胞中对一氧化氮（NO）表现出显著的响应，从而诱导Ca^<2+>进入。通过检测TRPC5半胱氨酸突变体对NO的反应性和s -亚硝基化的易感性，发现Cys553和Cys558是NO敏感性所必需的修饰位点。响应性TRPs在孔区相同的n端侧含有保守的半胱氨酸。引人注目的是，通过Cys553修饰的反应性二硫化物诱导的激活的膜侧性表明细胞质对Cys553的可及性。因此，基于化学生物学和生理学的多学科方法确定了TRP通道中NO敏感激活门的结构基元，建立了“NO传感器”作为扩展到不同TRP亚家族的新功能类别。