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

1.我们发现，IP 3结合1型IP 3受体（IP 3R）的“核心”区域位于225 - 578 a.a.的N-末端区域，并且在该区域内，至少三个碱性氨基酸（Arg-265、Lys-508、Arg-511）对于结合带负电荷的IP 3分子是必需的。在Sf 9细胞中表达的1型和3型的IP 3结合活性显示出与增加的Ca 2+相反的依赖性; 1型中对IP 3的亲和力降低（EC 50 = 100 nM Ca 2+），而3型中增加（872 nM）。IP 3诱导的Ca 2+释放（IICR）中，3型的IP 3敏感性低于1型（EC 50 = 358 nM vs 226 nM IP 3）。这些表明，胞浆Ca 2+以及aw IP 3浓度参与类型特异性IICR。2型被认为是“高亲和力”的IP 3R，然而，1型基因敲除小鼠的肝脏IP 3R（其中2型占优势）在IICR中显示出与1.4型相似的IP 3敏感性。通过在非洲爪蟾卵母细胞中表达与绿色荧光蛋白（GFP）的融合蛋白 关于我们 s，我们发现表达的C-末端区域包括一个假定的通道区域足以用于ER定位和四聚体的形成.建立了1型基因启动子控制的β-gal基因转基因小鼠品系，通过检测神经系统中β-gal活性分析了转基因小鼠的基因表达。我们在该启动子中鉴定了两个小脑特异性区域;一个是来自-398--295的阳性元件，另一个是来自-334--318的E-box样盒I。我们还测定了2型基因启动子的一级结构，发现存在多个转录起始位点.通过免疫组织化学方法，我们将1型IP 3R定位于血管平滑肌细胞和系膜细胞，2型定位于集合管的闰细胞，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型位于整个细胞质，而3型仅限于基底侧，这表明由于不同类型的亚细胞定位的极化的差异Ca 2+信号。少

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