Structural type 1 inositol 1,4,5-trisphosphate receptor

结构型 1 肌醇 1,4,5-三磷酸受体

• 批准号: 6857625
• 负责人: Irina I Serysheva
• 金额: $ 28.28万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-11 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6857625
• 关键词: SDS polyacrylamide gel electrophoresis; biochemistry; biological signal transduction; calcium channel; calcium ion; calmodulin; carbohydrate receptor; carbohydrate structure; conformation; cryoelectron microscopy; electrophysiology; inositol phosphates; laboratory rabbit; laboratory rat; membrane channels; membrane proteins; protein purification; western blottings

DESCRIPTION (provided by applicant): The inositol 1,4, 5 - trisphosphate receptors (IP3Rs) are large integral membrane proteins that function as the intracellular IP3-gated Ca 2+ release channels and govern rapid fluxes of Ca2+ ions from the endoplasmic reticulum into cytoplasm, thereby, playing a key role in a wide range of physiological functions including neurotransmitter release, fertilization, hormone secretion, gene transcription, metabolic regulation, apoptosis and muscle contraction. Abnormal regulation of Ca2+ homeostasis has been implicated in numerous human diseases such as cardiac hypertrophy, heart failure, hereditary ataxias, osteoporosis, atherosclerosis and some migraines. The long-term objectives of this project are to elucidate the molecular mechanisms of the IP3-induced Ca2+-gating through structure-function analysis of the IP3R channel complex and to define how defects in this channel protein can cause abnormal regulation of cell Ca2+ level underlying human diseases. The proposed project aims to utilize the electron cryomicroscopy and computer reconstruction techniques in conjunction with biochemical, electrophysiological, molecular and computational approaches to delineate the structural domains in the 3-D architecture of the IP3R1 (cerebellar isoform of IP3R) and to define structural steps underlying channel gating. The specific aims of this proposal are: 1) resolve the 3-D structure of the native IP3R1 in open and closed states; 2) ascertain topology of functional domains within quaternary structure of IP3R1; 3) elucidate the effects of calmodulin on the 3-D structure of the channel; 4) determine the 3-D structure of the recombinant constitutively open IP3RI. Proposed structural studies will exploit "single particles" approach, standing for isolated unordered particles. Thus, the purified IP3R1 channel particles will be trapped in different functional states by embedding in a thin layer of vitreous ice in the presence of channel specific modulators and then directly visualized in electron cryomicroscope. Sequence-specific antibodies will be employed to map regions of the primary sequence of the IP3R1, which are predicted to control intrinsic channel properties, in its 3-D structure. We anticipate that results from proposed studies will provide a three-dimensional framework for functional interpretations of the channel gating on which to base future biochemical, electrophysiological, and genetic experiments.

描述（由申请人提供）：肌醇1,4，5-三磷酸受体（IP3R）是大型的积分膜蛋白，作为细胞内IP3门控的Ca 2+释放通道的作用，并控制Ca2+离子的快速通量，从内质网中延伸到细胞群中，扮演较广泛的范围，扮演较广泛的角色，包括较广泛的功能，包括一项关键的角色，这些角色是一项关键的角色，这些角色既可以扮演关键的角色，又可以扮演关键的角色。受精，激素分泌，基因转录，代谢调节，凋亡和肌肉收缩。 CA2+稳态的异常调节与许多人类疾病有关，例如心脏肥大，心力衰竭，遗传性疾病，骨质疏松症，动脉粥样硬化和一些偏头痛。该项目的长期目标是通过对IP3R通道复合物的结构功能分析来阐明IP3诱导的Ca2+接种的分子机制，并定义该通道蛋白中的缺陷如何导致细胞CA2+水平的异常调节人类疾病。该提出的项目旨在利用电子冷冻机镜和计算机重建技术与生化，电生理学，分子和计算方法结合使用，以划定IP3R1的3-D结构中的结构域（IP3R的Cerebellar Isoform of IP3R），并在结构上进行结构步骤。该提案的具体目的是：1）在开放状态和封闭状态下解决本地IP3R1的3-D结构； 2）确定IP3R1第四纪结构内功能域的拓扑； 3）阐明钙调蛋白对通道3-D结构的影响； 4）确定重组组成型打开IP3RI的3-D结构。提出的结构研究将利用“单个颗粒”方法，代表孤立的无序颗粒。因此，纯化的IP3R1通道颗粒将在存在通道特异性调节剂的情况下嵌入薄薄的玻璃冰中，然后在电子冰冻显微镜中直接可视化。序列特异性抗体将用于映射IP3R1的主要序列的区域，该抗体被预测在其3-D结构中控制固有的通道特性。我们预计，拟议的研究的结果将为通道门控的功能解释提供一个三维框架，以基于未来的生化，电生理和遗传实验。