Pore architecture of the ORAI1 store-operated calcium channel.
ORAI1 is the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a store-operated channel that is central to Ca(2+) 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(2+). 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(2+) 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.
Zhou
Yubin
Y
Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital Boston, 200 Longwood Avenue, Boston, MA 02115, USA.
Ramachandran
Sweta
S
Oh-Hora
Masatsugu
M
Rao
Anjana
A
Hogan
Patrick G
PG
D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D000071740:ORAI1 Protein; D055503:Protein Multimerization / Q000187:drug effects; D017433:Protein Structure, Secondary
D015220:Calcium Channels; D003432:Cross-Linking Reagents; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C520430:Orai1 protein, mouse; D003545:Cysteine