Activation Mechanisms of Store-Operated Calcium Channels

 描述(申请人提供)：在许多动物细胞中，G蛋白或酪氨酸激酶偶联的细胞表面受体的刺激通过存储操作的钙释放激活的钙通道(CRAC)动员钙内流。随后的钙离子进入调节多种效应细胞的反应，包括转录、运动和增殖。CRAC通道显示出独特的生物物理指纹图谱，其特征是精致的钙离子选择性、存储操作的门控和独特的孔特性，是理解离子渗透和门控的生物物理机制的迷人的离子通道。此外，由于CRAC通道位于许多细胞中细胞内钙信号网络的连接点，CRAC通道功能的异常与多种疾病的病因有关，包括慢性炎症、肌肉无力和严重的联合免疫缺陷综合征。在过去的十年中，关于CRAC通道在不同组织中的生理作用以及CRAC通道激活过程的细胞编排已经学到了很多。尽管如此，内质网钙库枯竭如何调节CRAC通道孔开放的分子和结构基础仍然知之甚少。CRAC通道的开放是通过成孔的Orai蛋白和ER钙传感器STIM1之间的直接相互作用来控制的，但STIM1结合的转导如何影响Orai1通道孔的开放仍不清楚。在这里，我们提出了一种多管齐下的方法，将研究CRAC通道门控过程的几个机制方面，重点是CRAC通道孔中的分子重排和STIM1的构象变化。我们将为这些研究采用跨学科的实验设计，结合膜片钳电生理学、半胱氨酸突变、FRET显微镜、生化交联和蛋白质工程。利用Orai1蛋白作为CRAC通道的原型，我们的目标是：(1)测试CRAC通道孔的打开是通过TM1螺旋的旋转来进行的，从而重新定位疏水的V102侧链以消除能量障碍；(2)阐明Orai1通道的内部孔结构及其在调节Orai1通道离子传导中的作用；以及(3)研究STIM1在激活和与Orai1结合的过程中在至关重要的细胞质区域的构象重排。这些研究将极大地促进我们对CRAC通道激活的分子和结构机制的理解，并为利用CRAC通道的治疗潜力进行疾病干预开辟新的途径。

 DESCRIPTION (provided by applicant): In many animal cells, stimulation of cell surface receptors coupled to G proteins or tyrosine kinases mobilizes Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. The ensuing Ca2+ entry regulates a wide variety of effector cell responses including transcription, motility, and proliferation. CRAC channels exhibit a unique biophysical fingerprint characterized by exquisite Ca2+-selectivity, store-operated gating, and distinct pore properties, and serve as fascinating ion channels for understanding the biophysical mechanisms of ion permeation and gating. Moreover, because CRAC channels sit squarely at the nexus of the cellular Ca2+ signaling network in many cells, aberrant CRAC channel function is implicated in the etiology of several diseases including chronic inflammation, muscle weakness, and a severe combined immunodeficiency syndrome. Much has been learned in the last decade of the physiological roles of CRAC channels in different tissues and the cellular choreography of the CRAC channel activation process. Still, the molecular and structural basis of how depletion of ER Ca2+ stores regulates the opening of the CRAC channel pore continues to remain poorly understood. Opening of CRAC channels is governed through direct interactions between the pore-forming Orai proteins, and the ER Ca2+ sensor, STIM1, but how STIM1 binding transduces opening of the Orai1 channel pore remains unclear. Here, we propose a multi-pronged approach that will investigate several mechanistic aspects of the CRAC channel gating process, focusing on the molecular rearrangements in the CRAC channel pore and the conformational alterations in STIM1. We will employ an interdisciplinary experimental design for these studies that combines patch-clamp electrophysiology, cysteine mutagenesis, FRET microscopy, biochemical cross-linking, and protein engineering. Using the Orai1 protein as a prototypic CRAC channel, our goals are to: (1) test the hypothesis that opening of the CRAC channel pore occurs through rotation of the TM1 helix, thereby reorienting the hydrophobic V102 side-chains to remove an energy barrier, (2) elucidate the inner pore architecture and its role in regulating ion conduction in Orai1 channels, and (3) examine the conformational rearrangements in the critically important cytoplasmic region of STIM1 during activation and binding to Orai1. These studies will significantly advance our understanding of the molecular and structural mechanisms of CRAC channel activation and open new avenues for harnessing the therapeutic potential of CRAC channels for disease intervention.