Pore architecture of the ORAI1 store-operated calcium channel

Abstract

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.

Fig. 1.

Cysteineless ORAI1 used for the cross-linking studies. (A) Schematic depiction of the topology and primary sequence of cysteineless ORAI1. N-terminal residues 1–64 were replaced by a FLAG epitope, and the three native cysteine residues were replaced by valine residues (C126V/C143V/C195V, yellow circles). The glycosylation site was eliminated by an N223A substitution (pink circle). Residues E106 in TM1 and E190 in TM3, which have been linked to the Ca²⁺ selectivity of the channel, are highlighted in red. (B) Single-cell [Ca²⁺]_i measurements in primary CD4⁺ T cells. T cells (KO) had been infected with empty vector (magenta), expression vector for wild-type ORAI1 (green), or expression vector for cysteineless ORAI1 (red). T cells from a wild-type littermate (WT) received empty vector (black) or expression vector for cysteineless ORAI1 (blue). Intracellular Ca²⁺ stores were depleted by treatment with 1 μM thapsigargin (TG) in a nominally Ca²⁺-free buffer, and Ca²⁺ influx was monitored by the increase in cytoplasmic Ca²⁺ following readdition of extracellular Ca²⁺. Data are plotted as mean ± SEM.

Fig. 2.

E106C and TM1 are at an interface between ORAI1 monomers. (A–C) Membranes isolated from HEK293 cells expressing ORAI1 with a single cysteine residue in place of the TM1 residue indicated were incubated on ice for 10 min (A) in a buffer containing 0.3 mM DTT, (B) in a buffer supplemented with 1 mM CuP, or (C) in a buffer supplemented with 2 mM aqueous iodine (I₂). Samples were separated by SDS-PAGE under nonreducing conditions, and ORAI1 was detected by staining with anti-FLAG monoclonal antibody. (D Left) Cross-linking efficiency at each position is the fraction of total anti-FLAG staining in the dimer band. The range of the means in two experiments is plotted, except for A88C, L95C, and E106C, where the mean ± SEM for three experiments is given. (D Right) The cross-linking patterns of TM1 in incubations with CuP or with aqueous iodine are mapped onto the predicted α-helix.

Fig. 3.

Cross-linking of TM1 residues in a more extended CuP-catalyzed reaction. (A) Membranes containing ORAI1 with engineered cysteine residues at positions indicated were incubated on ice for 30 min with 10 mM CuP. Samples were separated by SDS-PAGE under nonreducing conditions, and ORAI1 was detected by staining with anti-FLAG monoclonal antibody. ORAI1 proteins with cysteine at positions showing efficient cross-linking in Fig. 2 were not included in this experiment. (B) Quantitation of cross-linking efficiency in the extended CuP-catalyzed reaction. Error bars represent the range of duplicate measurements.

Fig. 4.

E190C and surrounding TM3 residues fail to cross-link. (A) Analysis of ORAI1(E106C) and ORAI1(E190C) proteins incubated with CuP or with aqueous iodine under identical conditions. (B) Analysis of ORAI1 proteins with cysteine engineered into TM3 at the positions indicated. Treatment with 1 mM CuP or with aqueous iodine was carried out as in Fig. 2.

Fig. 5.

ORAI1 assembles as a tetramer or larger oligomer. (A) Membranes from HEK293 cells expressing the ORAI1 mutant A88C/V102C were subjected to cross-linking for 10 min on ice with the specified concentrations of CuP. (B) Membranes from HEK293 cells expressing the paired-cysteine mutants A88C/V102C, A88C/L95C, and L95C/V102C were subjected to cross-linking with 1 mM CuP for the specified periods on ice. (C) Diagram of cross-linking patterns that would produce covalently linked dimers, trimers, and tetramers. Only selected examples are shown.