冷冻超分辨光镜电镜融合解析钙释放激活钙通道细胞原位结构

Calcium release activated calcium (CRAC) channel is a highly selective plasma membrane Ca2+ ion channel gated by ER calcium depletion. CRAC functions in variety of cells in the regulation of Ca2+ concentration in the cytoplasm, dysfunction of which induce diseases in multi-systems in human. Basic functional components of CRAC complexes are ER Ca2+-sensor STIM1, and plasma membrane calcium channel protein ORAI1. Structural and functional studies have been intensely performed on these two membrane proteins. However, structural information on the stoichiometry of CRAC complexes are still lacking. Moreover, great controversy exists in the tetramer or hexamer composition of Orai1 Ca2+ channel due to the technical limitation of available methodologies on membrane proteins. Current study will employ self-established state-of-art cryogenic super-resolution Correlative Light and Electron microscopy (csCLEM) experimental systems to challenge a nano-scale structural analysis of CRAC complexes in situ on vitrified cellular context. The strategies we are employing for CRAC structural analysis would probe a new option to resolve the native structure of a membrane protein.

钙离子激活的钙（CRAC）通道是钙离子高度选择性的门控通道，是细胞精密调控胞质内钙离子浓度的重要途径。CRAC通道功能异常造成人体多系统疾病。CRAC复合体主要由ER钙离子浓度感受蛋白STIM1、以及质膜钙通道蛋白ORAI1组成。十几年来，人们对两个膜蛋白的结构功能进行了细致深入的研究。但CRAC复合体的基本分子组成信息仍旧匮乏，且膜通道Orai1蛋白的多聚体组装形式存在巨大争议。这主要是由现有膜蛋白研究技术的局限性导致的。本研究将应用前期建立的深冷超高分辨率光学显微镜融合冷冻三维电镜技术，在细胞原位解析生理状态下CRAC复合体的纳米尺度分子结构信息。我们在细胞原位解析CRAC复合体结构所做的技术摸索将为其它膜蛋白的结构解析提供新的思路和方法。