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Regulatory mechanism of intracellular Ca^<2+> dynamics

细胞内Ca^<2>动力学的调控机制

基本信息

批准号：
06044069
负责人：
MIKOSHIBA Katsuhiko
金额：
$ 5.12万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

The inositol 1,4,5-trisphosphate receptor (IP_3R) exisits as a tetrameric complex to form a functional inositol 1,4,5-trisphosphate-gated Ca^<2+> channel. Molecular cloning studies have shown that there are at least three types of IP_3R subunits, designated type 1, type 2, and type 3. The levels of expression of IP_3R subunits in various cell lines were investigated by Western blot analysis using type-specific antibodies against 15 C-terminal amino acids of each IP_3R subunits. We found that all the three types of IP_3R subunits were expressed in each cell line examined, but their levels of expression varied. To determine whether IP_3R from heterotetramers, we employed immunoprecipitation experiments using Chinese hamster ovary cells (CHO-K1 cells), in which all three types are abundantly expressed. Each type-specific antibody immunoprecipitated not only the respective cognate type but also the other two types. This result suggests that distinct types of IP_3R subunits assemble to form … More heterotetramers in CHO-K1 cells. We also detected heterotetramers in rat liver, in which IP_3R type 1 and type 2 are expressed abundantly. Previous studies have shown some functional differences among IP_3R types, suggesting the possibility that various compositions of submits show distinct channel properties. The diversity of IP_3R channels may be further increased by the co-assembly of different IP_3R subunits to form homo-or heterotetramers.Kinetics of Ca^<2+> release by adenophostin, a novel gaonist of inositol 1,4,5-trisphosphate (IP_3) receptor, in the purified and reconstituted IP_3 receptor type 1 (IP_3R1) was investigated using the fluorescent Ca^<2+> indicator fluo-3. Submaximal concentrations of adenophostin caused quantal Ca^<2+> release from the purified IP_3R1 as IP_3 did. Adenophostin-induced Ca^<2+> release by the purified IP_3R1 exhibited a high positive cooperativity (nH=3.9(]SY.+-.])0.2, EC_<50>=11 nM), whereas the IP_3-induced Ca^<2+> release exhibited a moderate one (nH=1.8(]SY.+-.])0.1, EC_<50>=1100 nM). Inhibitation of [^3H] IP_3 binding to the purified IP_3R1 by adenophostin exhibited a positive cooperativity (nH=1.9, K_i=10 nM), whereas IP_3 did not (nH=1.1, K_i=41 nM). Less

1，4，5-三磷酸肌醇受体（inositol 1，4，5-trisphosphate receptor，IP_3R）是一种四聚体复合物，可形成功能性的1，4，5-三磷酸肌醇门控Ca^2+通道。分子克隆研究表明，至少有三种类型的IP_3R亚基，命名为1型，2型和3型。利用针对每个IP_3R亚基C-末端15个氨基酸的类型特异性抗体，通过Western印迹分析来研究IP_3R亚基在各种细胞系中的表达水平。我们发现，所有三种类型的IP_3R亚基在每一个细胞系中检测表达，但它们的表达水平不同。为了确定IP_3R是否来自异源四聚体，我们使用中国仓鼠卵巢细胞（CHO-K1细胞）进行免疫沉淀实验，其中所有三种类型都大量表达。每种类型特异性抗体不仅免疫沉淀各自的同源类型，而且还免疫沉淀其他两种类型。这一结果表明，不同类型的IP_3R亚基组装形成 ...更多信息 CHO-K1细胞中的异源四聚体。我们还检测到异源四聚体在大鼠肝脏中，其中IP_3R1型和IP_3R2型表达丰富。以往的研究表明，IP_3R类型之间的一些功能差异，这表明不同的提交组合物显示不同的通道特性的可能性。用荧光Ca^2+指示剂Fluo-3研究了一种新的三磷酸肌醇（IP_3）受体激动剂腺苷（adenopropenin）对纯化和重组的IP_3受体1（IP_3R1）的Ca^2+释放动力学。亚最大浓度的腺苷可使纯化的IP_3R1产生与IP_3相同的Ca^<2+>量子释放。纯化的IP_3R1对Adenophostin诱导的Ca^<2+>释放具有很高的正协同效应（nH=3.9（[SY.+-.]）而<50>IP_3诱导的Ca^<2+>释放表现为中等水平（nH=1.8（]SY.+-.]）0.1，EC_<50>=1100 nM）。腺苷酸抑制[^3H] IP_3与纯化的IP_3R1的结合表现出正的协同效应（nH=1.9，Ki =10 nM），而IP_3则无此效应（nH=1.1，Ki =41 nM）。少