ORAI1 is the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a store-operated channel that is central to Ca 2þ signaling in mammalian cells. Electrophysiological data have shown that the acidic residues E106 in transmembrane helix 1 (TM1) and E190 in TM3 contribute to the high selectivity of ORAI1 channels for Ca 2þ . We have examined the pore architecture of the ORAI1 channel using ORAI1 proteins engineered to contain either one or two cysteine residues. Disulfide cross-linking shows that ORAI1 assembles as a tetramer or a higher oligomer with TM1 centrally located. Cysteine side chains projecting from TM1 at position 88, 95, 102, or 106 cross-link efficiently to the corresponding side chain in a second ORAI1 monomer. Cysteine residues at position 190 or at surrounding positions in TM3 do not cross-link. We conclude that E106 residues in wild-type ORAI1 are positioned to form a Ca 2þ binding site in the channel pore and that E190 interacts less directly with ions traversing the pore. The cross-linking data further identify a relatively rigid segment of TM1 adjacent to E106 that is likely to contribute to the selectivity filter.cross-linking | membrane protein | store-operated calcium entry | stromal interaction molecule | structure T he electrophysiologically defined calcium release-activated calcium (CRAC) channel is responsible for physiological Ca 2þ influx in T cells and mast cells and for store-operated Ca 2þ entry in other cells (1). CRAC currents in human T cells require the protein ORAI1 (2-4), which has been shown to be a plasma membrane Ca 2þ channel protein [(5-8); reviewed in refs. 9-11]. The severe combined immunodeficiencies that result from rare mutations in the human ORAI1 gene and the functional deficits of Orai1 −∕− mice dramatize the crucial signaling role played by ORAI1 in Tcells and mast cells (2,(12)(13)(14). The hallmarks of native CRAC channels and of recombinant ORAI1 channels are that they open in response to a reduction of Ca 2þ concentration in the endoplasmic reticulum (ER) lumen, that they are highly selective for Ca 2þ under physiological conditions, and that they carry only very small single-channel currents (1, 11). The sensitivity of the channel to the level of ER Ca 2þ stores has been traced to the ER-resident Ca 2þ sensor protein STIM1 (15)(16)(17)(18)(19)(20), but the basis for other channel properties is not understood in any detail.Point mutations introduced into the transmembrane helices of ORAI1 or its Drosophila orthologue alter ion selectivity (5-7). The replacements E106D or E190Q in human ORAI1 sharply reduce the ability of the channel to discriminate between Ca 2þ and Na þ under physiological conditions and reduce the decided preference of the channel for Na þ over Cs þ under conditions where the channel conducts monovalent ions (6, 7). However, although the electrophysiological analysis shows that E106 and E190 influence ion movements within the channel pore, it does not establish whether these residues do so directly by coordinating ...