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

描述（由申请人提供）：肌醇1,4,5 -三磷酸受体（IP3Rs）是一种大的整体膜蛋白，作为细胞内ip3门控的Ca2+释放通道，控制Ca2+离子从内质网快速流入细胞质，因此在广泛的生理功能中发挥关键作用，包括神经递质释放、生殖、激素分泌、基因转录、代谢调节、细胞凋亡和肌肉收缩。Ca2+体内平衡的异常调节与许多人类疾病有关，如心脏肥厚、心力衰竭、遗传性共济失调、骨质疏松、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-功能分析阐明ip3诱导的Ca2+门控的分子机制，并确定该通道蛋白的缺陷如何导致人类疾病中细胞Ca2+水平的异常调节。该项目旨在利用电子低温显微镜和计算机重建技术，结合生化、电生理、分子和计算方法来描绘IP3R1 （IP3R的小脑同型体）的三维结构域，并定义通道门控的结构步骤。本文的具体目标是：1)解析原生IP3R1在开放和封闭状态下的三维结构；2)确定IP3R1四元结构功能域的拓扑结构；3)阐明钙调素对通道三维结构的影响；4)确定重组本构开放IP3RI的三维结构。拟议的结构研究将利用“单粒子”方法，代表孤立的无序粒子。因此，纯化后的IP3R1通道粒子将在通道特异性调节剂存在的情况下，通过嵌入玻璃冰薄层，以不同的功能状态被捕获，然后在电子冷冻显微镜下直接可见。序列特异性抗体将用于绘制IP3R1初级序列的区域，这些区域被预测控制其内在通道特性，在其三维结构中。我们预计，这些研究的结果将为通道门控的功能解释提供一个三维框架，并以此为基础进行未来的生化、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.