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The structure and function of Ca^<2+>-permeable nonselective cation channels

Ca^2-渗透性非选择性阳离子通道的结构和功能

基本信息

批准号：
11670086
负责人：
MIWA Soichi
金额：
$ 2.3万
依托单位：
Hokkaido University (2000)Kyoto University (1999)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670086/
关键词：
endothelin-1 vascular smooth muscle cell nonselective cation channels store-operated Ca^<2+> channel SK＆F 96365 LOE 908 transient receptor potential カルシウムチャンネルクローニングストア作動性カルシウムチャンネルカルシウム

项目摘要

The purpose of the present study is to clarify the structure, function, pharmacology and functional significance of Ca^<2+> channels activated bv endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs). ET-1 activated three types of Ca^<2+>-permeable channel in A7r5 cells derived from rat thoracic aortic smooth muscle cells : two types of nonselective cation channel (designated NSCC-1 and NSCC-2) and store-operated Ca^<2+> channel (SOCC). Importantly, these channels were discriminated by two drugs like SK＆F 96365 and LOE 908 belonging to blockers of the "so-called" receptor-operated Ca^<2+> channel. Using these blockes, contractions and inreases in the intracellular free Ca^<2+> concentration induced by ET-1 in VSMCs were found to actually involve Ca^<2+> entry through NSCC-1, NSCC-2 and SOCC.To isolate cDNAs encoding these channels, we used two strategies : one is PCR-based cloning and the other is expression cloning with Xenopus oocytes. In the former strategy, the conserved region of Ca^<2+> channels called transient receptor potentials (trp) was used as probe. In the latter strategy, a screening method using ^<45>Ca^<2+> uptake was developed to specifically detect Ca^<2+> entry. In the PCR-based method, two forms of cDNA (trp 4 and its putative splice variant designated trp 4Δ) were isolated. The sequence of trp 4 was essentially similar to that isolated from other tissues, but trp 4Δ was unique in that it lacks part of the sequence corresponding to the first and second membrane spanning region. When expressed in the oocytes and HEK 293 cells, both cDNAs were functionally intact. They were activated by store depletion, and showed the same pharmacological properties as those of SOCC.Notably, trp 4Δ showed larger responses than trp 4. In the expression cloning strategy, cDNA library from VSMCs was screened and five positive clones were obtained. The structure and function of these clones are being determined.

本研究的目的是阐明血管平滑肌细胞(VSMCs)内皮素-1(ET-1)激活的钙通道的结构、功能、药理作用和功能意义。ET-1在大鼠胸主动脉平滑肌细胞A7r5细胞上激活三种钙离子通透性通道：非选择性阳离子通道(NSCC-1和NSCC-2)和钙离子通道(SOCC)。重要的是，这些通道被SK&F 96365和LOE 908这两种药物区分开来，这两种药物属于“所谓的”受体操作的钙通道阻滞剂。利用这些阻滞剂，我们发现ET-1引起的VSMC内游离钙离子浓度的升高和收缩实际上涉及到通过NSCC-1、NSCC-2和SOCC进入细胞内。为了分离编码这些通道的cDNAs，我们采用了两种策略：一种是基于PCR的克隆，另一种是利用非洲爪哇卵母细胞进行表达克隆。在前一种策略中，以钙通道的保守区称为瞬时受体电位(Trp)作为探针。在后一种策略中，开发了一种使用^&lt；45&gt；Ca^&lt；2+&gt；摄取的筛选方法来特异性地检测Ca^&lt；2+&gt；条目。在基于聚合酶链式反应的方法中，分离到两种形式的cDNATrP4及其可能的剪接变异体TrP4Δ。Trp 4的序列与从其他组织中分离到的序列基本相似，但Trp 4的Δ是独一无二的，因为它缺少与第一和第二膜跨越区域相对应的部分序列。当在卵母细胞和HEK 293细胞中表达时，这两个cDNA在功能上是完整的。其药理活性与SOCC相同。值得注意的是，Trp-4的Δ活性高于Trp-4。这些克隆的结构和功能正在确定中。