Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.
Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.
复制标题
DOI：
10.1126/scisignal.aad6281
复制
发表时间：
2016-04-05
影响因子：
7.3
通讯作者：
Yule DI
中科院分区：
文献类型：
--
作者：
Alzayady KJ;Wang L;Chandrasekhar R;Wagner LE 2nd;Van Petegem F;Yule DI
肌醇1，4，5-三磷酸（IP 3）受体（IP 3Rs）是四聚体的细胞内Ca 2+释放通道，每个亚基在N-末端含有IP 3的结合位点。我们提供的证据表明，四个IP 3分子需要在不同的条件下激活通道。比较结合和Ca 2+释放的浓度-响应关系表明，IP 3Rs在大量Ca 2+释放发生之前被IP 3最大限度地占据。我们发现，当与鸡免疫细胞系DT 40 - 3 KO中的野生型单体共表达时，配体结合缺陷亚基以显性负性方式发挥作用，该细胞系缺乏编码IP 3R亚基的所有三种基因，并在通过CRISPR/Cas9技术产生的IP 3R无效人类细胞系（HEK-3 KO）中证实了相同的效果。使用二聚体和四聚体级联IP 3R与越来越多的结合缺陷亚基，我们解决了专性配体的化学计量。具有四个配体结合位点的串联IP 3Rs表现出天然IP 3Rs的Ca 2+释放和电生理特性。然而，IP 3未能激活IP 3Rs组装从级联二聚体组成的一个结合能力和一个结合缺陷的突变亚基。类似地，含有两个IP 3R 2短单体（IP 3结合缺陷剪接变体）的IP 3R是无功能的。串联四聚体只含有三个有结合能力的配体结合位点，在广泛的活化条件下是无功能的。这些数据提供了明确的证据，即IP 3诱导的Ca 2+释放仅发生在四聚体内的每个IP 3R单体被IP 3占据时，从而确保Ca 2+释放的保真度。
Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca2+-release channels with each subunit containing a binding site for IP3 in the N-terminus. We provide evidence that four IP3 molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca2+ release suggested that IP3Rs are maximally occupied by IP3 before substantial Ca2+ release occurs. We showed that ligand binding–deficient subunits acted in a dominant-negative manner when coexpressed with wild-type monomers in the chicken immune cell line DT40-3KO, which lacks all three genes encoding IP3R subunits, and confirmed the same effect in an IP3R-null human cell line (HEK-3KO) generated by CRISPR/Cas9 technology. Using dimeric and tetrameric concatenated IP3Rs with increasing numbers of binding-deficient subunits, we addressed the obligate ligand stoichiometry. The concatenated IP3Rs with four ligand-binding sites exhibited Ca2+ release and electrophysiological properties of native IP3Rs. However, IP3 failed to activate IP3Rs assembled from concatenated dimers consisting of one binding-competent and one binding-deficient mutant subunit. Similarly, IP3Rs containing two monomers of IP3R2short, an IP3 binding-deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding competent ligand-binding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP3-induced Ca2+ release only occurs when each IP3R monomer within the tetramer is occupied by IP3, thereby ensuring fidelity of Ca2+ release.