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

描述(申请人提供)：肌醇1，4，5-三磷酸受体(IP3Rs)是一种大的整膜蛋白，作为细胞内IP3门控的钙释放通道，控制钙离子从内质网快速流入细胞质，从而在神经递质释放、受精、激素分泌、基因转录、代谢调节、细胞凋亡和肌肉收缩等广泛的生理功能中发挥关键作用。钙稳态调节异常与多种人类疾病有关，如心肌肥厚、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。该项目的长期目标是通过对IP3R通道复合体的结构和功能的分析，阐明IP3诱导的钙门控的分子机制，并确定该通道蛋白的缺陷如何导致与人类疾病相关的细胞钙水平的异常调节。该项目旨在利用电子冷冻显微镜和计算机重建技术，结合生化、电生理、分子和计算方法来描绘IP3R1(IP3R的小脑异构体)三维结构中的结构域，并确定通道门控的结构步骤。这一建议的具体目的是：1)解析天然IP3R1在开放和关闭状态下的三维结构；2)确定IP3R1四级结构中功能结构域的拓扑结构；3)阐明钙调蛋白对通道三维结构的影响；4)确定重组结构性开放IP3RI的三维结构。拟议的结构研究将采用“单一粒子”的方法，代表孤立的无序粒子。因此，在通道特异性调节剂存在的情况下，通过将纯化的IP3R1通道颗粒嵌入薄层玻璃冰中，纯化的IP3R1通道颗粒将被捕获在不同的功能状态，然后在电子冷冻显微镜中直接可视化。序列特异性抗体将被用来定位IP3R1的初级序列的区域，这些区域被预测控制其三维结构中的固有通道属性。我们预计，拟议的研究结果将为通道门控的功能解释提供一个三维框架，以此为基础进行未来的生化、电生理和遗传学实验。

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.