Molecular characterization of the multi-modal regulation of inositol 1,4,5-trisphosphate receptors

项目总结 本项目的目标是表征多模式配体的分子机制-- 1，4，5-三磷酸肌醇受体(IP3Rs)依赖门控。IP3R无处不在地表达 内质网(ER)钙通道在维持钙稳态中起关键作用 将内质网中储存的钙离子传导到细胞质中。在胞浆中，许多受钙调节的蛋白质感觉 局部和整体钙浓度的变化以调节不同的细胞过程，包括受精、细胞 死亡和分化。由于胞质钙调控过程的多样性，IP3R介导 钙离子的电导受到离子、小分子和蛋白质辅助因子的精确调节。两个初选 IP3Rs的调节配体是肌醇1，4，5-三磷酸(IP3)和细胞质钙，两者都结合 用于调制气孔的选通状态的通道。而IP3结合激活IP3Rs、胞浆钙 在低浓度时激活通道，在高浓度时抑制通道。两相性 钙离子与IP3R活性的关系保证了细胞质中的钙离子浓度适当 受监管的。除了IP3和钙外，IP3R活性还由其他小分子以细胞特异性的方式进一步塑造 分子，如三磷酸腺苷，由翻译后修饰IP3Rs的酶和许多蛋白质共同作用 各种因素。如何综合这些不同因素的影响来确定IP3R门控状态，从而 在分子水平上对细胞内钙信号的调控仍然知之甚少。AS对IP3R活性的失调 与心脏疾病、癌症、神经紊乱和其他病理有关，了解调节 IP3R将与人类健康和疾病具有广泛的相关性。 该提案旨在利用结构、生化和生物物理方法来开发一种机械 了解IP3R的调控，重点是钙离子、IP3和Bcl2家族的三个蛋白辅助因子：BCL-2， MCL-1和Bc l-xl。通过这些方法，我们的目标是了解i)IP3和钙结合是如何共同稳定的 活性构象中的IP3R，II)过量的钙是如何抑制离子传导的，III)蛋白质是如何协同作用的 BCL-2、MCL-1和BCL-XL在IP3和Ca~(2+)存在的情况下调节通道活动。拟议的研究将 揭示多个刺激如何整合以调节离子通道功能的原理。由于广博 IP3R的生理作用，这些研究得出的发现将与许多领域相关 包括离子通道、钙信号、细胞死亡和神经生物学。 好了！

PROJECT SUMMARY The objective of this project is to characterize the molecular mechanisms of the multi-modal ligand- dependent gating of inositol 1,4,5-trisphosphate receptors (IP3Rs). IP3Rs are ubiquitously expressed endoplasmic reticulum (ER) Ca2+ channels that play a key role in the maintenance of Ca2+ homeostasis by conducting Ca2+ stored in the ER into the cytoplasm. In the cytosol, numerous Ca2+-regulated proteins sense changes in local and global Ca2+ concentrations to regulate diverse cellular process including fertilization, cell death and differentiation. Because of the diversity of processes regulated by cytoplasmic Ca2+, IP3R-mediated Ca2+ conductance is precisely regulated by ions, small molecules and protein co-factors. The two primary regulatory ligands of IP3Rs are inositol 1,4,5-trisphosphate (IP3) and cytoplasmic Ca2+, both of which bind the channel to modulate the gating state of the pore. Whereas IP3 binding activates IP3Rs, cytoplasmic Ca2+ both activates the channel at low concentrations and inhibits the channel at high concentrations. The biphasic relationship between Ca2+ and IP3R activity ensures that cytoplasmic Ca2+ concentrations are properly regulated. Besides IP3 and Ca2+, IP3R activity is further shaped in cell-specific manner by other small molecules such as ATP, by enzymes that post-translationallly modify IP3Rs and by numerous protein co- factors. How the effects of these various factors are synthesized to determine IP3R gating state and thus regulate cellular Ca2+ signalling remain poorly understood at a molecular level. As dysregulation of IP3R activity is linked to cardiac disease, cancer, neurological disorders and other pathologies, understanding the regulation of IP3Rs will have broad relevance to human health and disease. The proposal aims to employ structural, biochemical and biophysical approaches to develop a mechanistic understanding of IP3R regulation, focusing on Ca2+, IP3 and three protein co-factors of the Bcl-2 family: Bcl-2, Mcl-1 and Bcl-xL. With these approaches we aim to understand i) how IP3 and Ca2+ binding jointly stabilize IP3Rs in an active conformation, ii) how excess Ca2+ inhibits ion conduction and iii) how the protein co-factors Bcl-2, Mcl-1 and Bcl-xL modulate channel activity in the presence of IP3 and Ca2+. The proposed studies will reveal principles of how multiple stimuli are integrated to regulate ion channel function. Due to the broad physiological role of IP3Rs, the finding derived from these studies will be relevant to a number of fields including ion channels, Ca2+ signaling, cell death and neurobiology. !