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

项目概要 该项目的目的是表征多模式配体的分子机制 肌醇 1,4,5-三磷酸受体 (IP3R) 的依赖性门控。 IP3R 无处不在 内质网 (ER) Ca2 通道在维持 Ca2 稳态中发挥关键作用 将储存在 ER 中的 Ca2 传导到细胞质中。在细胞质中，许多 Ca2 调节蛋白感知 局部和整体 Ca2 浓度的变化来调节多种细胞过程，包括受精、细胞 死亡和分化。由于细胞质 Ca2 调节过程的多样性，IP3R 介导 Ca2 电导受到离子、小分子和蛋白质辅助因子的精确调节。两个主要 IP3R 的调节配体是肌醇 1,4,5-三磷酸 (IP3) 和细胞质 Ca2 ，两者均与 通道来调节孔的门控状态。而 IP3 结合会激活 IP3R，细胞质 Ca2 低浓度时激活通道，高浓度时抑制通道。双相 Ca2 和 IP3R 活性之间的关系确保细胞质 Ca2 浓度正确 受监管。除了 IP3 和 Ca2 之外，IP3R 活性还通过其他小分子以细胞特异性方式进一步形成。 诸如 ATP 等分子，通过翻译后修饰 IP3R 的酶以及大量的蛋白质辅酶 因素。如何综合这些不同因素的影响来确定 IP3R 门控状态，从而确定 IP3R 门控状态 调节细胞 Ca2 信号传导在分子水平上仍知之甚少。由于 IP3R 活性失调 与心脏病、癌症、神经系统疾病和其他病症有关，了解其调节 IP3R 与人类健康和疾病具有广泛的相关性。 该提案旨在采用结构、生物化学和生物物理方法来开发机械 了解 IP3R 调节，重点关注 Ca2、IP3 和 Bcl-2 家族的三个蛋白质辅因子：Bcl-2、 Mcl-1 和 Bcl-xL。通过这些方法，我们的目标是了解 i) IP3 和 Ca2 结合如何共同稳定 处于活性构象的 IP3R，ii) 过量的 Ca2 如何抑制离子传导，以及 iii) 蛋白质如何辅助因子 Bcl-2、Mcl-1 和 Bcl-xL 在 IP3 和 Ca2 存在的情况下调节通道活性。拟议的研究将 揭示如何整合多种刺激来调节离子通道功能的原理。由于广泛 IP3R 的生理作用，这些研究的发现将与许多领域相关 包括离子通道、Ca2 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. !