Store-operated calcium entry through the plasma membrane CRAC channel in mammalian T cells and mast cells depends on the sensor protein STIM1 and the channel subunit ORAI1.ORAI1 is the pore-forming subunit of the CRAC channel that permits calcium influx through the plasma membrane upon emptying of internal calcium stores.Electrophysiological data have suggested that the acidic residue El06 in transmembrane helix 1 (TM1) forms an essential binding site for calcium ions in the channel pore.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 positions 88, 95, 102, or 106 cross-link efficiently to the corresponding side chain in a second ORAI1 monomer.Thus protein biochemical and electrophysiological evidence independently indicate that El06 residues in wild-type ORAI1 are positioned to form a calcium binding site in the channel pore.The cross-linking data further identify a relatively rigid nonpolar segment of TM1 just internal to E106 that is likely to contribute to the selectivity filter, and a flexible segment of TM1 that adjoins the N-terminal STIM1-binding site implicated in channel gating.