Structure and function of inositol triphosphate receptors

项目总结 1，4，5-三磷酸肌醇受体(IP3Rs)整合了激素、生长发育等多种信号。 因子、神经递质和代谢状态的变化来调节所有细胞类型中的下游信号。 IP3R是受变构和共价机制进一步调节的配体门控离子通道， 介导内质网(ER)的钙释放。由此产生的细胞质和 线粒体钙浓度调节许多生理过程(例如，学习、记忆、膜 运输、突触传递、分泌、运动性、膜兴奋性、基因表达、细胞分裂和 细胞凋亡)。此外，IP3Rs和钙信号的病理性失调与癌症有关， 神经退行性疾病、自身免疫疾病和代谢性疾病，使IP3Rs成为治疗 这些疾病。尽管最近在结构研究方面取得了进展，但关于 配体相互作用和通道门控的机制大多仍未得到解答，部分原因是 IP3R的规模和复杂性以及特定药理工具的可获得性有限。 在这个提案中，我们将(目标1)结合低温电子显微镜(Cryo-EM)和X射线结晶学 结合基于荧光的钙成像的功能IP3R分析来阐明 IP3R门控循环的一般主题和小分子抑制受体的分子基础。 我们最近公布的数据显示，IP3结合位点被我们称为 自结合多肽(SBP)，位于初级序列的远端。我们假设 SBP是IP3Rs中的一个新的调节位点，可以调节对IP3的表观亲和力，从而调节钙离子 通道活动，以及IP3R亚型之间SBP序列的差异有助于其不同的 监管属性。我们将对IP3R亚型和SBP进行(目标2)功能和结构研究 突变体来检验这一假说，并确定这种相互作用的结构决定因素。 完成这些目标将产生对IP3R门槛和监管的无与伦比的机械性洞察， 潜在地导致了IP3Rs新的和特定的药理调节剂的开发。此外 这些化合物将作为人们长期寻求的研究工具来研究IP3R，将作为一个起点 用于开发新的治疗方法来治疗与IP3R活性异常相关的疾病。

PROJECT SUMMARY Inositol 1,4,5-triphosphate receptors (IP3Rs) integrate diverse signals generated by hormones, growth factors, neurotransmitters, and changes in metabolic state to modulate downstream signaling in all cell types. IP3Rs are ligand-gated ion channels that are further regulated by allosteric and covalent mechanisms, mediating Ca2+ release from the endoplasmic reticulum (ER). The resulting increases of cytoplasmic and mitochondrial Ca2+ concentrations regulate many physiological processes (e.g., learning, memory, membrane trafficking, synaptic transmission, secretion, motility, membrane excitability, gene expression, cell division, and apoptosis). Furthermore, pathological dysregulation of IP3Rs and calcium signaling is implicated in cancer, neurodegenerative, autoimmune, and metabolic diseases, making IP3Rs promising targets for treatment of these diseases. Despite recent advances in structural studies, fundamental questions regarding the mechanisms of ligand interactions and channel gating remain mostly unanswered, in part because of the large size and complexity of IP3Rs and the limited availability of specific pharmacological tools. In this proposal, we will (Aim 1) combine cryo-electron microscopy (cryo-EM) and X-ray crystallography in conjunction with functional IP3R assays based on fluorescence-based calcium imaging to elucidate the general themes of IP3R gating cycle and molecular basis for receptor inhibition by small molecules. Our recently published data revealed that the IP3 binding site is occupied by a loop that we have termed the self-binding peptide (SBP), which is located distantly in the primary sequence. We hypothesize that the SBP is a novel regulatory site in IP3Rs that can modulate the apparent affinity for IP3, and thereby Ca2+ channel activity, and that the divergence of SBP sequences between IP3R subtypes contributes to their distinct regulatory properties. We will perform (Aim 2) functional and structural studies on IP3R subtypes and SBP mutants to test this hypothesis and identify the structural determinants of this interaction. Completion of these aims will yield unparalleled mechanistic insight into IP3R gating and regulation, potentially leading to the development of novel and specific pharmacological modulators of IP3Rs. In addition to being used as a long-sought research tools to study IP3Rs, these compounds will serve as a starting point for development of novel therapeutic approaches to treat diseases associated with aberrant IP3R activity.