Regulation of inositol trisphosphate receptors

钙是一种控制多种细胞反应的通用信使，包括肌肉收缩， 胞吐、记忆、受精、新陈代谢、增殖和细胞死亡。大量荷尔蒙， 神经递质和生长因子刺激肌醇1，4，5-三磷酸(IP3)的形成 位于内质网的受体家族(IP3Rs)作为配体门控钙离子的功能 频道。细胞内钙库的耗尽也激活了血浆中的钙内流机制 薄膜。因此，IP3与其受体的相互作用激活了钙信号的所有阶段。IP3R是 受钙离子和磷酸化的调节，但这种调节的分子基础尚不清楚。CA2 从IP3Rs释放出来的物质局部传递到线粒体，可以刺激新陈代谢，在更高的 量多了，也会引发细胞死亡。癌细胞被认为高度依赖IP3R- 介导的线粒体钙离子转移为它们的生存。CRISPR/CAS-9技术的出现使得 HEK293和HeLa宫颈癌细胞三种IP3R亚型的基因消融该提案是 以这些细胞系的使用为中心a)研究允许细胞在 完全没有钙信号，以及b)利用结构-功能的零背景 IP3开放通道的分子机制和反馈机制的研究 由钙离子调节。两个具体目标是：1]表征IP3R-3KO细胞的适应机制： 缺乏钙调节新陈代谢的影响将通过测量几种生物能量来调查 参数和~(13)C示踪剂代谢。增殖、细胞周期状态、细胞死亡和转录重新连接 将会被探索。2A]IP3是如何打通频道的？将使用化学交联/质谱学 验证冷冻-EM结构并监测IP3在天然IP3R中介导的构象变化。 关键残基的突变将被用来测试拟议的门控变构机制。2B]识别 IP3R中的钙调节位点：基于IP3R1和IP3R3的最新低温EM结构 已经确定了3簇带负电荷的残基，这些残基是钙离子调控位点的候选者。他们的 作为刺激或抑制部位的功能作用将通过突变来评估。这些网站的作用将是 也可以通过诱变辣椒果实中对钙离子不敏感的IP3R进行检测。长期目标 为了更好地理解钙信号在正常细胞、癌细胞和遗传性细胞中的作用 紊乱使IP3R功能失活。研究还应提供有关以下方面的基本机制信息 这些重要渠道是如何运作和受到监管的。

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.