Structure-function studies of IP3R channels

描述（由申请人提供）：Ca2+通过IP3R通道释放是最普遍和最通用的细胞信号机制，在多种生理功能的调节中发挥关键作用，包括受精，激素分泌，基因转录，代谢调节，免疫反应，细胞凋亡，学习和记忆。尽管IP3Rs在生理和病理上具有重要意义，但这些通道在天然和疾病状态下的功能的分子机制仍然知之甚少，主要是因为缺乏关于IP3Rs 3D结构的高分辨率结构细节。考虑到IP3Rs的大尺寸（~1.3 MDa），它们在膜环境中的位置以及它们的动态性质，这种结构被证明是非常难以获得的。本研究的重点是1型IP3R (IP3R1)，这是小脑浦肯野细胞中ip3门控Ca2+释放通道的主要类型。迄今为止，整个IP3R1的最佳结构是用中分辨率（10-15¿）的单粒子电子冷冻显微镜（cryo-EM）分辨出来的，晶体结构仅限于细胞质区域的可溶部分，仅占整体结构的约15%。因此，围绕IP3R通道门控的大多数关键问题仍然是模糊的。在本应用中，我们试图回答有关IP3R1门控的基本问题：Ca2+通过IP3R1渗透的结构决定因素是什么，配体如何控制门控过程以及IP3R通道中孔隙打开的构象变化。为了解决这些问题，我们将整个IP3R1的结构确定扩展到亚纳米分辨率，并将在接近天然脂质环境中确定其结构。我们将结合冷冻电镜，计算工具和生物信息学的结构方法与生物化学和生物物理技术，包括放射性配体结合，基于荧光的Ca2+通量测定和脂质双分子层通道记录。这种多学科方法将允许将结构分析与通道功能相关联。提出的结构研究将利用单粒子低温电镜方法。因此，IP3R1蛋白复合物将从清洁剂溶解的微粒体膜中纯化出来，并以嵌入玻璃体冰的单个颗粒的形式可见。IP3R1的结构将在载脂蛋白（目标1）和配体结合状态（目标2）下进行分析，方法是在体外重现功能相关条件，并在EM网格上冻结捕获反应。然后，我们建议将IP3R1通道重构为小的单层脂质囊泡，并使用单颗粒重构的变体来解决脂质膜中的通道结构（目标3）。随着这些研究的完成，我们期望建立IP3R1功能的结构和机制基础，并阐明调节通道门控的分子机制缺陷如何导致细胞Ca2+水平异常，从而导致许多疾病。

DESCRIPTION (provided by applicant): Ca2+ release via IP3R channels is the most ubiquitous and versatile cellular signaling mechanism that plays a key role in the regulation of diverse physiological functions, including fertilization, hormone secretion, gene transcription, metabolic regulation, immune responses, apoptosis, learning and memory. Despite established significance of IP3Rs in physiology and pathology, the molecular mechanisms underlying function of these channels, both in native and disease states, remain poorly understood, mainly because of the lack of high- resolution structural details about the 3D architecture of IP3Rs. Such structures have proven exceptionally difficult to obtain given the large size of IP3Rs (~1.3 MDa), their location in the membrane environment, and their dynamic nature. The focus of this proposal is type 1 IP3R (IP3R1), the predominant type of IP3-gated Ca2+ release channel in cerebellar Purkinje cells. To date, the best structure of the entire IP3R1 is resolved by single-particle electron cryomicroscopy (cryo-EM) at intermediate resolution (10-15 ¿), and the crystal structures are limited to a soluble portion of the cytoplasmic region representing only ~15% of the overall structure. Therefore, most critical issues surrounding gating of IP3R channels are stil ambiguous. In this application, we seek to answer the fundamental questions on IP3R1 gating: what are the structural determinants of Ca2+ permeation through IP3R1, how ligands control the gating process and what conformational changes underlie pore opening in IP3R channels. To address these questions, we will extend structure determination of the entire IP3R1 to sub-nanometer resolution and will determine its structure in a near-native lipid environment. We will combine structural methodologies from cryo-EM, computational tools and bioinformatics with biochemical and biophysical techniques including radioligand binding, fluorescence- based Ca2+ flux assays and lipid bilayer channel recordings. This multidisciplinary approach will allow correlating structural analysis with channel function. Proposed structural studies will exploit single-particle cryo- EM methodology. Thus, the IP3R1 protein complex will be purified from detergent solubilized microsomal membranes and visualized in the form of individual particles embedded in vitreous ice. The IP3R1 structure will be analyzed in Apo- (aim 1) and ligand-bound states (aim 2) by reproducing the functionally relevant conditions in vitro and freeze-trapping the reaction on the EM grid. We then propose to reconstitute IP3R1 channel into small unilamellar lipid vesicles and to use a variant of single-particle reconstruction to solve the channel structure in the lipid membrane (aim 3). With these studies accomplished, we anticipate to establish the structural and mechanistic basis for IP3R1 function and to elucidate how defects in molecular mechanisms regulating the channel's gating can lead to abnormal cell Ca2+ levels underlying numerous diseases.