Regulatory mechanism of intracellular Ca^<2+> dynamics

肌醇1,4,5-三磷酸受体（IP_3R）以四聚体复合物形式存在，形成功能性肌醇1,4,5-三磷酸门控Ca^<2+>通道。分子克隆研究表明，至少有三种类型的IP_3R亚基，指定类型1，类型2和类型3。Western blot检测IP_3R亚基在不同细胞系中的表达水平，并对每个IP_3R亚基的15个c端氨基酸进行型特异性抗体检测。我们发现所有三种类型的IP_3R亚基在每个细胞系中都有表达，但它们的表达水平不同。为了确定IP_3R是否来自异源四聚体，我们使用中国仓鼠卵巢细胞（CHO-K1细胞）进行了免疫沉淀实验，这三种类型的细胞都大量表达。每种类型特异性抗体不仅免疫沉淀各自的同源型，而且免疫沉淀其他两种类型。这一结果表明，不同类型的IP_3R亚基在CHO-K1细胞中聚集形成更多的异源四聚体。我们还在大鼠肝脏中检测到异源四聚体，其中IP_3R 1型和2型大量表达。先前的研究表明IP_3R类型之间存在一些功能差异，这表明不同的提交物组成可能表现出不同的通道特性。IP_3R通道的多样性可以通过不同IP_3R亚基的共组装形成同质或异质四聚体而进一步增加。采用荧光Ca^<2+>指示剂fluo-3研究了一种新型肌醇1,4,5-三磷酸（IP_3）受体腺磷酸腺苷（adenophostin）在纯化重组的IP_3受体1 （IP_3R1）中释放Ca^<2+>的动力学。与IP_3一样，亚最大浓度的腺磷酸导致纯化的IP_3R1释放大量Ca^<2+>。纯化的IP_3R1诱导的Ca^<2+>释放具有较高的正协同性（nH=3.9(]SY.+- -])0.2, EC_<50>=11 nM），而ip_3诱导的Ca^<2+>释放具有中等的正协同性（nH=1.8(]SY.+- -])0.1, EC_<50>=1100 nM）。腺宿主抑制[^3H] IP_3与纯化的IP_3R1的结合表现出正的协同性（nH=1.9, K_i=10 nM），而IP_3则没有（nH=1.1, K_i=41 nM）。少

The inositol 1,4,5-trisphosphate receptor (IP_3R) exisits as a tetrameric complex to form a functional inositol 1,4,5-trisphosphate-gated Ca^<2+> channel. Molecular cloning studies have shown that there are at least three types of IP_3R subunits, designated type 1, type 2, and type 3. The levels of expression of IP_3R subunits in various cell lines were investigated by Western blot analysis using type-specific antibodies against 15 C-terminal amino acids of each IP_3R subunits. We found that all the three types of IP_3R subunits were expressed in each cell line examined, but their levels of expression varied. To determine whether IP_3R from heterotetramers, we employed immunoprecipitation experiments using Chinese hamster ovary cells (CHO-K1 cells), in which all three types are abundantly expressed. Each type-specific antibody immunoprecipitated not only the respective cognate type but also the other two types. This result suggests that distinct types of IP_3R subunits assemble to form … More heterotetramers in CHO-K1 cells. We also detected heterotetramers in rat liver, in which IP_3R type 1 and type 2 are expressed abundantly. Previous studies have shown some functional differences among IP_3R types, suggesting the possibility that various compositions of submits show distinct channel properties. The diversity of IP_3R channels may be further increased by the co-assembly of different IP_3R subunits to form homo-or heterotetramers.Kinetics of Ca^<2+> release by adenophostin, a novel gaonist of inositol 1,4,5-trisphosphate (IP_3) receptor, in the purified and reconstituted IP_3 receptor type 1 (IP_3R1) was investigated using the fluorescent Ca^<2+> indicator fluo-3. Submaximal concentrations of adenophostin caused quantal Ca^<2+> release from the purified IP_3R1 as IP_3 did. Adenophostin-induced Ca^<2+> release by the purified IP_3R1 exhibited a high positive cooperativity (nH=3.9(]SY.+-.])0.2, EC_<50>=11 nM), whereas the IP_3-induced Ca^<2+> release exhibited a moderate one (nH=1.8(]SY.+-.])0.1, EC_<50>=1100 nM). Inhibitation of [^3H] IP_3 binding to the purified IP_3R1 by adenophostin exhibited a positive cooperativity (nH=1.9, K_i=10 nM), whereas IP_3 did not (nH=1.1, K_i=41 nM). Less