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

项目摘要 该项目的目标是表征多模态配体的分子机制- 1，4，5-三磷酸肌醇受体（IP 3R）的依赖性门控。IP 3R普遍表达于 内质网（ER）Ca 2+通道，在维持Ca 2+稳态中起关键作用， 将内质网中储存的Ca 2+导入细胞质。在胞质溶胶中，许多Ca 2+调节的蛋白质感测 局部和整体Ca 2+浓度的变化，以调节不同的细胞过程，包括受精，细胞 死亡与分化由于细胞质Ca 2+调节的过程的多样性，IP 3R介导的 Ca 2+电导由离子、小分子和蛋白质辅因子精确调节。两个主要 IP 3R的调节配体是肌醇1，4，5-三磷酸（IP 3）和细胞质Ca 2+，两者都结合细胞质中的Ca 2+。 通道以调节孔的门控状态。而IP 3结合激活IP 3Rs，细胞质Ca 2+都 在低浓度下激活通道，在高浓度下抑制通道。双相 Ca 2+和IP 3R活性之间的关系确保了细胞质Ca 2+浓度适当 监管.除了IP 3和Ca 2+外，IP 3R活性还通过其他小的细胞因子以细胞特异性方式进一步形成。 分子，如ATP，通过酶后修饰IP 3R和许多蛋白质共- 因素如何综合这些不同因素的影响来确定IP 3R门控状态，从而 调节细胞Ca 2+信号传导在分子水平上仍然知之甚少。由于IP 3R活性失调， 与心脏病、癌症、神经系统疾病和其他病理学有关， 知识产权3R将对人类健康和疾病产生广泛的影响。 该提案旨在采用结构，生物化学和生物物理方法来开发一种机制， 了解IP 3R调节，重点是Ca 2+，IP 3和Bcl-2家族的三种蛋白质辅因子：Bcl-2， Mcl-1和Bcl-xL。通过这些方法，我们的目标是了解i）IP 3和Ca 2+结合如何共同稳定 活性构象中的IP 3R，ii）过量的Ca 2+如何抑制离子传导和iii）蛋白质辅因子如何 Bcl-2、Mcl-1和Bcl-xL在IP 3和Ca 2+存在下调节通道活性。拟议的研究将 揭示了多种刺激如何整合以调节离子通道功能的原理。由于宽 IP 3R的生理作用，这些研究的结果将与许多领域有关 包括离子通道、Ca 2+信号传导、细胞死亡和神经生物学。 !

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. !