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

肌醇1,4,5-三磷酸（IP_3）是磷脂酰肌醇4,5-二磷酸在胞外刺激激活质膜上的G蛋白或酪氨酸激酶偶联受体时水解产生的第二信使。利用ip3r内在缺陷的DT40细胞作为受体表达的宿主细胞，我们试图建立一个能够进行重组ip3r受体（ip3r）功能分析的实验系统。通过对140多个细胞系的筛选，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表明外源性表达的IP_3R1与天然IP_3Rs一样定位于内质网。B细胞受体刺激可诱导表达IP_3R1的小鼠细胞质Ca^<2+>升高，表明重组IP_3R1在稳定细胞系中功能正常。将这些细胞系制备的膜组分重组成平面脂质双层后，我们检测到了二价阳离子电流。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。在目前的表达系统中，IP_3R的成功功能表达为我们提供了一个详细的IP_3R通道结构-功能表征的机会。

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.