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

描述（申请人提供）：1，4，5 -三磷酸肌醇受体（inositol 1，4，5 - trisphosphate receptor，IP 3Rs）是一种重要的细胞膜蛋白，是细胞内IP 3门控的Ca 2+释放通道，控制Ca 2+从内质网向细胞质的快速流动，在神经递质释放、受精、激素分泌、基因转录、代谢调节、细胞凋亡和肌肉收缩。Ca 2+稳态的异常调节与许多人类疾病有关，如心脏肥大、心力衰竭、遗传性共济失调、骨质疏松症、动脉粥样硬化和一些偏头痛。本项目的长期目标是通过IP 3R通道复合物的结构-功能分析阐明IP 3诱导的Ca 2+门控的分子机制，并确定该通道蛋白的缺陷如何导致细胞Ca 2+水平的异常调节，从而导致人类疾病。该项目旨在利用电子冷冻显微镜和计算机重建技术，结合生物化学，电生理学，分子和计算方法来描绘IP 3R 1（IP 3R的小脑亚型）的3-D架构中的结构域，并定义通道门控的结构步骤。该方案的具体目标是：1）解析天然IP 3R 1在开放和闭合状态下的3-D结构; 2）确定IP 3R 1四级结构内的功能结构域的拓扑结构; 3）阐明钙调蛋白对通道的3-D结构的影响; 4）确定重组组成型开放IP 3RI的3-D结构。拟议的结构研究将利用“单粒子”的方法，代表孤立的无序粒子。因此，纯化的IP 3R 1通道颗粒将通过在通道特异性调节剂的存在下包埋在玻璃状冰的薄层中而以不同的功能状态被捕获，然后在电子低温显微镜中直接可视化。序列特异性抗体将被用于映射IP 3R 1的一级序列的区域，其被预测为控制内在通道特性，在其3-D结构中。我们预计，从拟议的研究结果将提供一个三维框架的通道门控功能的解释基础上，未来的生化，电生理和遗传实验。

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.