Regulation of inositol trisphosphate receptors

钙是一种通用的信使，控制着包括肌肉收缩在内的多种细胞反应， 胞吐、记忆、受精、代谢、增殖和细胞死亡。无数的荷尔蒙， 神经递质和生长因子刺激肌醇1，4，5-三磷酸（IP 3）的形成， 位于内质网中的一个受体家族（IP 3R），作为配体门控Ca 2 + 渠道细胞内Ca 2+储存的耗尽也激活了血浆中的Ca 2+内流机制 膜的因此，IP 3与其受体的相互作用激活了Ca 2+信号的所有阶段。IP 3R是 受Ca 2+和磷酸化调节，但这种调节的分子基础知之甚少。Ca2+ 从IP 3Rs释放的蛋白质局部传递到线粒体，可以刺激新陈代谢，在较高的 量，也可以引发细胞死亡。已经提出癌细胞高度依赖于IP 3R。 介导的线粒体Ca 2+转移对它们的存活起重要作用。CRISPR/Cas-9技术的出现， 来自HEK 293和HeLa宫颈癌细胞的所有三种IP 3R同种型的遗传消除。该提案 集中在这些细胞系的使用，以a）研究允许细胞在环境中存活的适应机制， 完全不存在Ca 2+信号传导和B）利用结构-功能的空背景 探索IP 3开放通道的分子机制和反馈机制的研究 Ca 2+的调节。两个具体目标是：1]表征IP 3R-3 KO细胞中的适应机制： 将通过测量几种生物能量来研究缺乏Ca 2+调节代谢的影响。 参数和13 C示踪剂代谢。增殖、细胞周期状态、细胞死亡和转录重连 将被探索。2A] IP 3如何打开通道？将使用化学交联/质谱法 以验证cryo-EM结构并监测天然IP 3R中由IP 3介导的构象变化。 关键残基的突变将用于检测拟定的门控变构机制。2B]鉴别 IP 3R中的Ca 2+调节位点：基于IP 3R 1和IP 3R 3的最新cryo-EM结构， 已经鉴定了3簇带负电荷的残基，它们是Ca 2+调节位点的候选者。他们的 作为刺激或抑制位点的功能作用将通过诱变来评估。这些网站的作用将 还通过诱变来自Capsaspora owczarzaki的Ca 2 +-不敏感的IP 3R来测试。远景目标 该提案的目的是更好地了解Ca 2+信号在正常细胞，癌细胞和遗传性疾病中的作用。 使IP 3R功能失活的病症。这项研究还应提供基本的机械信息， 这些重要的渠道是如何运作和管理的。

Calcium is a universal messenger controlling a multitude of cellular responses including muscle contraction, exocytosis, memory, fertilization, metabolism, proliferation and cell death. Numerous hormones, neurotransmitters and growth factors stimulate the formation of inositol 1,4,5-trisphosphate ( IP3 ) which acts on a family of receptors ( IP3Rs ) located in the endoplasmic reticulum that function as ligand-gated Ca2+ channels. The depletion of intracellular Ca2+ stores also activates Ca2+ influx mechanisms in the plasma membrane. Thus, the interaction of IP3 with its receptor activates all phases of a Ca2+ signal. IP3Rs are regulated by Ca2+ and phosphorylation but the molecular basis of this regulation is poorly understood. Ca2+ released from IP3Rs is locally transmitted to the mitochondria and can stimulate metabolism, and in higher amounts, can also initiate cell death. Cancer cells have been proposed to be highly dependent on IP3R- mediated mitochondrial Ca2+ transfer for their survival. The advent of CRISPR/Cas-9 technology has allowed the genetic ablation of all three IP3R isoforms from HEK293 and HeLa cervix carcinoma cells. The proposal is centered on the use of these cell lines to a) study the adaptive mechanisms allowing the cells to survive in the complete absence of Ca2+ signaling and b) to take advantage of a null background for structure-function studies exploring the molecular mechanism by which IP3 opens the channel and the mechanism of feed-back regulation by Ca2+. The two specific aims are: 1] Characterize adaptive mechanisms in IP3R-3KO cells: The impact of a lack of Ca2+ regulation of metabolism will be investigated by measuring several bioenergetic parameters and 13C-tracer metabolism. Proliferation, cell-cycle status, cell death and transcriptional rewiring will be explored. 2A] How does IP3 open the channel? Chemical crosslinking/mass spectroscopy will be used to validate cryo-EM structures and monitor conformational changes mediated by IP3 in native IP3Rs. Mutagenesis of key residues will be used to test proposed allosteric mechanisms of gating. 2B] Identification of the Ca2+ regulatory sites in IP3Rs: Based on the most recent cryo-EM structures of IP3R1 and IP3R3 we have identified 3 clusters of negatively charged residues that are candidates for Ca2+ regulatory sites. Their functional role as stimulatory or inhibitory sites will be assessed by mutagenesis. The role of these sites will also be tested by mutagenesis of the Ca2+-insensitive IP3R from Capsaspora owczarzaki . The long-term goal of the proposal is to better understand the role of Ca2+signaling in normal cells, cancer cells and in inherited disorders inactivating IP3R function. The study should also provide fundamental mechanistic information on how these important channels work and are regulated.