Study of the Molecular Structure and Function of IP_3 Receptor/Ca^<2+> Release Channel

IP_3受体/Ca^<2>释放通道的分子结构与功能研究

基本信息

批准号：
08459009
负责人：
FURUICHI Teiichi
金额：
$ 6.08万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08459009/
关键词：
IP3 receptor ligand binding second messenger calcium channel ion channel calcium release intracellular calcium inositol 1,4,5-trisphosphate

项目摘要

1. We found that the "core" region for IP3 binding in the type 1 IP3 receptor (IP3R) is located in the N-terminal region from 225 to 578 a.a., and that within this region, at least three basic amino acids (Arg-265, Lys-508, Arg-511) are essential for binding to the negatively-charged IP3 molecule.2. The IP3 binding activity of type 1 and 3 expressed in Sf9 cells displayd an opposite dependency to increasing Ca2+ ; affinity for IP3 is decreased in type 1 (EC50=100nM Ca2+), whereas increased in type 3 (872nM). IP3 sensitivity in IP3-induced Ca2+ release (IICR) of type 3 was lower than that of type 1 (EC50=358nM vs 226nM IP3). These suggest that cytosolic Ca2+ as well aw IP3 concentrations are involved in type-specific IICR.3. Type 2 has been thought to be "high affinity" IP3R.However, liver IP3R of type 1 knock-out mice, in which type 2 predominates, showed a similar IP3 sensitivity in IICR as type 1.4. By expressing a fusion protein with green fluorescent protein (GFP) in Xenopus oocyte … More s, we showed that the expressed C-terminal region including a putative channel region is sufficient for the ER localization and the tetramer formation.5. We established transgenic mouse lines carrying beta-gal gene controlled by type 1 gene promoter, and analyzed the gene expression by detecting beta-gal activity in the nervous system. We identified two cerebellum-specific regions in this promoter ; one is a positive element from -398--295, the other is an E-box-like box I from -334--318. We also determined the primary structure of type 2 gene promoter, and found the presence of multiple transcription initiation sites.6. By immunohistochemistry of rat kidney, we localized type 1 IP3R to vascular smooth muscle cells and mesangial cells, type 2 to intercalated cells of the collecting duct, and type 3 to vascular smooth muscle cells, mesangial cells and principal cells of the cortical collecting duct. Intracellularly, type 1 and 2 are located throughout the cytoplasm, whereas type 3 is restricted to the basolateral side, suggesting the differential Ca2+ signaling due to the polarization in the subcellular localization of distinct types. Less

1。我们发现1型IP3受体（IP3R）中IP3结合的“核心”区域位于N末端区域，从225到578 A.A.，并且在该区域内，至少三个碱性氨基酸（ARG-265，LYS-508，ARG-511）对于与负收率的IP3 Molecule.2是必不可少的。在SF9细胞中表达的1型和3的IP3结合活性表现出与CA2+增加的相反的依赖性。 IP3的亲和力在1型（EC50 = 100nm Ca2+）中有所下降，而3型（872nm）的增长率下降。 IP3诱导的3型Ca2+释放（IICR）中的IP3灵敏度低于1型（EC50 = 358nm vs 226nm IP3）。这些表明胞质Ca2+以及AW IP3浓度涉及特异性IICR.3。 2型被认为是“高亲和力” IP3R。但是，类型1基因敲除小鼠的肝IP3R，其中2型占主导地位，在IICR中显示出类似于1.4类型的IP3敏感性。通过在异爪蟾卵母细胞中用绿色荧光蛋白（GFP）表达融合蛋白……更多S，我们表明，包括推定通道区域的表达的C末端区域足以用于ER定位和Tetramer组。5。我们建立了由1型基因启动子控制的β-GAL基因的转基因小鼠系，并通过检测神经系统中的β-GAL活性来分析基因表达。我们确定了该启动子中的两个小脑特异性区域。一个是-398--295的积极元素，另一个是-334---318的类似电子盒的盒子I。我们还确定了2型基因启动子的主要结构，并发现了多个转录起始位点的存在。6。通过大鼠肾脏的免疫组织化学，我们将1型IP3R局部定位于血管平滑肌细胞和弥赛亚细胞，2型到收集导管的插入细胞，以及3型的血管平滑肌细胞，弥赛亚和皮质收集管的主要细胞。细胞内，1型和2型均位于整个细胞质中，而3型则限于基层侧，这表明由于不同类型的亚细胞定位的极化而引起的差异Ca2+信号传导。较少的