权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Physiological significance of feedback regulation of inositol 1,4,5-trisphosphate receptor by cytoplasmic calcium

细胞质钙对肌醇1,4,5-三磷酸受体反馈调节的生理意义

基本信息

批准号：
12680652
负责人：
MICHIKAWA Takayuki
金额：
$ 2.24万
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 (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

The inositol 1,4,5-trisphosphate (IP_3) is a second messenger that is produced by hydrolysis of phosphatidyl-inositol 4,5-bisphosphate in response to activation by extracellular stimuli of the G protein- or tyrosine-kinase-coupled receptors on the plasma membrane. Using intrinsically IP_3R-deficient DT40 cells as the host cells for the receptor expression, we have tried to set up an experimental system in which functional analysis of recombinant IP_3 receptors (IP_3Rs) is practicable. After screening over 140 cell lines, we have established eleven stable cell lines that express mouse type 1 IP_3R (IP_3R1) with various contents. Both fluorescence immunocytochemical analysis and immunoelectron microscopic analysis showed that exogenously expressed IP_3R1 is localized on endoplasmic reticulum as native IP_3Rs. Stimulation of B cell receptor induced cytoplasmic Ca^<2+> increases in the mouse IP_3R1-expressing cells, indicating that the recombinant IP_3R1 in the stable cell lines functions properly. After reconstitution of membrane fractions prepared from these cell lines into planar lipid bilayers, we detected divalent cation currents. Single channel conductance, IP_3-dependency, and cytoplasmic Ca^<2+> dependency of the observed currents were similar to those of native IP_3R channels. Successful functional expression of IP_3R in the present expression system provides us an opportunity for detailed structure-functional characterization of IP_3R channels.

肌醇 1,4,5-三磷酸 (IP_3) 是第二信使，是通过响应质膜上 G 蛋白或酪氨酸激酶偶联受体的细胞外刺激而水解磷脂酰肌醇 4,5-二磷酸而产生的。使用本质上IP_3R缺陷的DT40细胞作为受体表达的宿主细胞，我们试图建立一个实验系统，在该系统中重组IP_3受体（IP_3Rs）的功能分析是可行的。经过筛选超过140个细胞系，我们建立了11个表达不同含量的小鼠1型IP_3R（IP_3R1）的稳定细胞系。荧光免疫细胞化学分析和免疫电镜分析均表明外源表达的IP_3R1与天然IP_3R一样定位于内质网。 B细胞受体的刺激诱导表达小鼠IP_3R1的细胞中细胞质Ca 2+ 增加，表明稳定细胞系中的重组IP_3R1正常发挥作用。将这些细胞系制备的膜组分重构为平面脂质双层后，我们检测到二价阳离子电流。观察到的电流的单通道电导、IP_3依赖性和细胞质Ca 2+ 依赖性与天然IP_3R通道相似。 IP_3R 在本表达系统中的成功功能表达为我们提供了对 IP_3R 通道进行详细结构功能表征的机会。