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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端区域225至578个氨基酸，并且在该区域内，至少有3个碱性氨基酸（Arg-265、Lys-508、Arg-511）对于与带负电荷的IP3分子的结合是必需的。2. Sf9 细胞中表达的 1 型和 3 型 IP3 结合活性表现出与增加 Ca2+ 相反的依赖性； 1 型中 IP3 的亲和力降低 (EC50=100nM Ca2+)，而 3 型中 IP3 的亲和力增加 (872nM)。 3型的IP3诱导Ca2+释放(IICR)的IP3敏感性低于1型(EC50=358nM vs 226nM IP3)。这些表明胞质 Ca2+ 以及 aw IP3 浓度与类型特异性 IICR.3 有关。 2型被认为是“高亲和力”IP3R。然而，1型敲除小鼠（其中2型占主导地位）的肝脏IP3R在IICR中表现出与1.4型相似的IP3敏感性。通过在非洲爪蟾卵母细胞中表达带有绿色荧光蛋白（GFP）的融合蛋白，我们发现表达的C端区域（包括假定的通道区域）足以用于内质网定位和四聚体形成。5．我们建立了携带由1型基因启动子控制的β-gal基因的转基因小鼠品系，并通过检测神经系统中的β-gal活性来分析基因表达。我们在该启动子中鉴定了两个小脑特异性区域；一个是-398--295的正元素，另一个是-334--318的类似E盒的盒子I。我们还确定了2型基因启动子的一级结构，发现存在多个转录起始位点。 6．通过大鼠肾脏的免疫组织化学，我们将1型IP3R定位于血管平滑肌细胞和系膜细胞，2型定位于集合管的嵌入细胞，3型定位于血管平滑肌细胞、系膜细胞和皮质集合管的主细胞。在细胞内，1 型和 2 型遍布整个细胞质，而 3 型仅限于基底外侧，这表明由于不同类型的亚细胞定位中的极化而导致不同的 Ca2+ 信号传导。较少的