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. 2009 Dec 29;106(52):22516-21.
doi: 10.1073/pnas.0909574106. Epub 2009 Dec 11.

Structural determinants of ion permeation in CRAC channels

Beth A McNally  1 , Megumi Yamashita, Anita Engh, Murali Prakriya
Affiliations

Structural determinants of ion permeation in CRAC channels

Beth A McNally et al. Proc Natl Acad Sci U S A. .

Abstract

CRAC channels generate Ca(2+) signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca(2+) selectivity, low Cs(+) permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiol-reactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca(2+) selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd(2+) by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Primary structure and predicted topology of Orai1. (A) Important residues identified from structure-function and human linkage-analysis studies are highlighted. The red boxes indicate the regions of the protein targeted for Cys scanning in this study. (B) Amino acid sequence alignment of the three human Orai isoforms showing conserved residues (shaded) in TM1, the TM1-TM2 loop, and TM3.
Fig. 2.
Fig. 2.
Cysteine substitutions at some positions form inter-subunit disulfide bonds. (A) Application of BMS (5 mM) immediately following whole-cell break-in enhances ICRAC in Q108C Orai1. (B) Western blot of lysates from cells expressing STIM1+Q108C Orai1 reveals Orai1 dimers. Exposing the lysate to increasing concentrations β-mercaptoethanol dose-dependently decreases the dimer:monomer protein ratio, confirming disulfide cross-linking between Q108C Orai1 subunits. (C) Western blots of several Orai1 Cys mutants reveal bands corresponding to dimers in E106C, Q108C, D110C, A111C, and D112C Orai1 mutants. (D) The pattern of Orai1 mutants exhibiting disulfide cross-linking is similar in the Cys-less Orai1 background. All cells were treated with tunicamycin to eliminate protein glycosylation (Fig. S3A).
Fig. 3.
Fig. 3.
Covalent modification of Orai1 Cys mutants by MTS reagents. (A and B) WT and Cys-less Orai1 channels are insensitive to modification by MTSET. In both cases, ICRAC (measured at −100 mV) was partially inhibited by application of MTSET (100 μM), but this inhibition reversed completely following washout of the reagent. BMS (5 mM) treatment transiently inhibited ICRAC by an unknown mechanism. (C) Modification of D110C Orai1 (in the Cys-less background) by MTSET, MTSEA, and MTS-TEAE (100 μM each) recorded from the same cell. In each case, significant persistent inhibition is seen following washout of the reagent that is reversed by BMS (5 mM). Voltage ramps in the right graph show the I-Vs at the time points indicated by arrowheads.
Fig. 4.
Fig. 4.
MTS reagents block Cys mutants localized exclusively in the TM1-TM2 loop. (A) Summary of persistent inhibition [100 * (1 − Ipost/Ipre), where Ipre and Ipost are the currents prior and post washout of the MTS reagent, respectively] of single-Cys mutants in the WT Orai1 background given as mean (±SEM; n = 4–12 cells). WTO1, wild-type Orai1. Asterisks indicate mutations that produced nonfunctional channels. (B) Pattern of block of the mutants in the Cys-less background by MTSET and MTS-TEAE. Positions V107 to D110 are highly reactive to MTSET both in the WT and Cys-less backgrounds, indicating minimal contributions of native cysteines to the observed reactivity. CLO1, Cys-less Orai1. (C) MTSET reaction rate constants of the most susceptible mutants (mean ± SEM of four cells). (D) MTSES does not produce persistent blockade of D110C currents, but eliminates subsequent persistent blockade by MTSET. Persistent MTSET block is restored following treatment with BMS, which presumably removes MTSES. The right graph shows the mean ± SEM (n = 4) persistent block by MTSET applied at the time points indicated in the left trace.
Fig. 5.
Fig. 5.
Cd2+ block reveals pore-lining residues in TM1 (A) Blockade of ICRAC in various Cys mutants by 5 μM Cd2+ (WT background). Although Cd2+ block in V102C and L95C was poorly reversed by washout, application of a chelator-containing DVF solution (V102C) or the reducing agent BMS (L95C) reversed Cd2+ block. (B) Pattern of Cd2+ blockade (5 μM) of TM1 and TM1-TM2 loop Cys mutants (±SEM; n = 4–12 cells). In the TM1 segment, three residues (L95C, G98C, and V102C) exhibited strong reactivity to Cd2+. The dotted line depicts a sine function with a periodicity of 3.5. Asterisks indicate mutations that produced nonfunctional channels. (C and D) E190C and the other TM3 positions (WT background) do not exhibit significant sensitivity to Cd2+ (5 μM). (E) Rate constants of Cd2+ blockade of selected TM1 and TM1-TM2 loop Cys mutants in WT background (±SEM; n = 4 cells). (F) The most Cd2+ sensitive sites in TM1 (green boxes) localize on one face of a helical wheel, suggesting that this segment forms a α helical structure. E106C (red) is nonfunctional. R91C (yellow box) exhibited only modest reactivity.
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