Ma Guolin, He Lian, Jing Ji, Tan Peng, Huang Yun, Zhou Yubin
Center for Translational Cancer Research, Institute of Biosciences and Technology, College of Medicine, Texas A&M University, 2121 W. Holcombe Blvd, Houston, TX, USA.
Department of Medical Physiology, College of Medicine, Texas A&M University, Temple, TX, USA.
Methods Mol Biol. 2018;1843:147-166. doi: 10.1007/978-1-4939-8704-7_13.
ORAI1 constitutes the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a prototypical store-operated channel that is essential for the activation of cells of the immune system. Here we describe a convenient yet powerful cross-linking approach to examine the pore architecture of CRAC channels using ORAI1 proteins engineered to contain one or two cysteine residues. The generalizable cross-linking in situ approach can also be readily extended to study other integral membrane proteins expressed in various types of cells.
ORAI1构成了钙释放激活钙（CRAC）通道的成孔亚基，CRAC通道是一种典型的储存操纵性通道，对免疫系统细胞的激活至关重要。在此，我们描述了一种简便而强大的交联方法，用于使用经工程改造含有一个或两个半胱氨酸残基的ORAI1蛋白来研究CRAC通道的孔结构。这种可推广的原位交联方法也可以很容易地扩展到研究在各种类型细胞中表达的其他整合膜蛋白。
