Abstract
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.