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

肌醇1,4,5-三磷酸受体（IP_3R）作为四聚体络合物存在，形成功能性肌醇1,4,5-三磷酸磷酸盐门控的Ca^<2+>通道。分子克隆研究表明，指定的IP_3R亚基至少有三种类型的IP_3R亚基，指定类型1，类型2和类型3。通过使用蛋白质印迹分析，使用针对每种IP_3R亚基的每种IP_3R亚基的15 c-末端氨基酸的类型抗体，通过蛋白质印迹分析研究了各种细胞系中IP_3R亚基的表达水平。我们发现，所有三种类型的IP_3R亚基都在检查的每个细胞系中表达，但它们的表达水平各不相同。为了确定IP_3R是否来自异源次聚体的IP_3R，我们使用了中国仓鼠卵巢细胞（CHO-K1细胞）进行免疫沉淀实验，其中所有三种类型都绝对表达。每种类型的特异性抗体免疫沉淀不仅为CHO-K1细胞中的更多异晶剂。我们还检测到大鼠肝脏中的异驱动器，其中IP_3R类型1和类型2彻底表示。先前的研究表明，IP_3R类型之间的一些功能差异表明，各种提交的组合物显示出不同的信道特性的可能性。 IP_3R通道的多样性可以通过不同的IP_3R亚基的共同组装来进一步增加，以形成同型或异光学器。Ca^<2+>通过腺粘蛋白释放的基础化学，使用含量为1,4,4,5-Trisosphate（IP_3）受体的IP_3）的含量的高含量（IP_3）受体IP_3的IPSITOR，IP_3）的含量（ip_3）受体。荧光Ca^<2+>指示器Fluo-3。像IP_3一样，腺植物的次最大浓度导致纯化的IP_3R1释放量化ca^<2+>。通过纯化的IP_3R1释放腺苷蛋白诱导的Ca^<2+>暴露了高阳性协调（NH = 3.9（]SY。+ - 。]）0.2，EC_ <50> = 11 nm），而IP_3诱导的Ca^<2+>则释放了一个中等的一个（NH = 1.8（NH = 1.8（] SYM）。抑制[^3H] IP_3通过腺植物抑制纯化的IP_3R1结合，暴露了正配位（NH = 1.9，K_I = 10 nm），而IP_3则没有（NH = 1.1，K_I，K_I = 41 nm）。较少的