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STIM1 triggers a gating rearrangement at the extracellular mouth of the ORAI1 channel.

Gudlur A, Quintana A, Zhou Y, Hirve N, Mahapatra S, Hogan PG - Nat Commun (2014)

Bottom Line: We show that interaction of STIM1 with the cytoplasmic face of the human ORAI1 channel elicits a conformational change near the external entrance to the pore, detectable at the pore Ca(2+)-binding residue E106 and the adjacent pore-lining residue V102.We demonstrate that a short nonpolar segment of the pore including V102 forms a barrier to ion flux in the closed channel, implicating the STIM1-dependent movement in channel gating.Our data explain the close coupling between ORAI1 channel gating and ion selectivity, and open a new avenue to dissect the gating, modulation and inactivation of ORAI-family channels.

View Article: PubMed Central - PubMed

Affiliation: Division of Signalling and Gene Expression, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037, USA.

ABSTRACT
The ER-resident regulatory protein STIM1 triggers store-operated Ca(2+) entry by direct interaction with the plasma membrane Ca(2+) channel ORAI1. The mechanism of channel gating remains undefined. Here we establish that STIM1 gates the purified recombinant ORAI1 channel in vitro, and use Tb(3+) luminescence and, separately, disulfide crosslinking to probe movements of the pore-lining helices. We show that interaction of STIM1 with the cytoplasmic face of the human ORAI1 channel elicits a conformational change near the external entrance to the pore, detectable at the pore Ca(2+)-binding residue E106 and the adjacent pore-lining residue V102. We demonstrate that a short nonpolar segment of the pore including V102 forms a barrier to ion flux in the closed channel, implicating the STIM1-dependent movement in channel gating. Our data explain the close coupling between ORAI1 channel gating and ion selectivity, and open a new avenue to dissect the gating, modulation and inactivation of ORAI-family channels.

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STIM1-mediated conformational change at residue V102(a) Tb3+ luminescence from ORAI1V102C showing STIM1CT (S1CT)-dependent enhancement of the Tb3+ signal comparable to that observed with ORAI1WT (Figure 2b). Representative of 3 experiments. (b,c) Western blot analyses of S. cerevisiae sec6-4 vesicles expressing the Flag-tagged ORAI proteins V102C ORAI1 and LSK/V102C ORAI1, following Cu2+-phenanthroline crosslinking in the absence or presence of STIM1CT. Multiple samples were treated in parallel for each condition. Red and black arrowheads demarcate ORAI1 dimer and monomer bands, respectively. Representative of 3–5 experiments. (d) Fraction of ORAI1 in the dimer band (mean ± SEM) from gels shown in Figure 5b–c. ****, p < 0.0001 by two-tailed Student’s t-test. No change was found in disulfide-linked dimer formation of FLAG-ORAI1LSK/V102C following STIM1CT treatment (NS, p > 0.05).
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Figure 5: STIM1-mediated conformational change at residue V102(a) Tb3+ luminescence from ORAI1V102C showing STIM1CT (S1CT)-dependent enhancement of the Tb3+ signal comparable to that observed with ORAI1WT (Figure 2b). Representative of 3 experiments. (b,c) Western blot analyses of S. cerevisiae sec6-4 vesicles expressing the Flag-tagged ORAI proteins V102C ORAI1 and LSK/V102C ORAI1, following Cu2+-phenanthroline crosslinking in the absence or presence of STIM1CT. Multiple samples were treated in parallel for each condition. Red and black arrowheads demarcate ORAI1 dimer and monomer bands, respectively. Representative of 3–5 experiments. (d) Fraction of ORAI1 in the dimer band (mean ± SEM) from gels shown in Figure 5b–c. ****, p < 0.0001 by two-tailed Student’s t-test. No change was found in disulfide-linked dimer formation of FLAG-ORAI1LSK/V102C following STIM1CT treatment (NS, p > 0.05).

Mentions: The Cu2+-phenanthroline crosslinking assay that we developed to map ORAI1 pore architecture (44) is an independent way to probe for a conformational change at the external entrance to the pore. Treatment with Cu2+-phenanthroline crosslinks pairs of cysteine side chains that are positioned suitably and is very sensitive to the local geometry of protein side chains, able to discriminate, for example, between the ‘a’ and ‘d’ positions in the hydrophobic core of a coiled coil (45). We focused on V102, whose side chain projects into the pore one helical turn below E106. The valine > cysteine substitution needed for the crosslinking assay is conservative, replacing the valine side chain with a slightly smaller side chain of comparable hydrophobicity, and we verified that both the initial Tb3+ luminescence signal of the V102C channel and the conformational change reported by a STIM-induced increase in Tb3+ luminescence resemble those of the wildtype channel (Figure 5a).


STIM1 triggers a gating rearrangement at the extracellular mouth of the ORAI1 channel.

Gudlur A, Quintana A, Zhou Y, Hirve N, Mahapatra S, Hogan PG - Nat Commun (2014)

STIM1-mediated conformational change at residue V102(a) Tb3+ luminescence from ORAI1V102C showing STIM1CT (S1CT)-dependent enhancement of the Tb3+ signal comparable to that observed with ORAI1WT (Figure 2b). Representative of 3 experiments. (b,c) Western blot analyses of S. cerevisiae sec6-4 vesicles expressing the Flag-tagged ORAI proteins V102C ORAI1 and LSK/V102C ORAI1, following Cu2+-phenanthroline crosslinking in the absence or presence of STIM1CT. Multiple samples were treated in parallel for each condition. Red and black arrowheads demarcate ORAI1 dimer and monomer bands, respectively. Representative of 3–5 experiments. (d) Fraction of ORAI1 in the dimer band (mean ± SEM) from gels shown in Figure 5b–c. ****, p < 0.0001 by two-tailed Student’s t-test. No change was found in disulfide-linked dimer formation of FLAG-ORAI1LSK/V102C following STIM1CT treatment (NS, p > 0.05).
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Figure 5: STIM1-mediated conformational change at residue V102(a) Tb3+ luminescence from ORAI1V102C showing STIM1CT (S1CT)-dependent enhancement of the Tb3+ signal comparable to that observed with ORAI1WT (Figure 2b). Representative of 3 experiments. (b,c) Western blot analyses of S. cerevisiae sec6-4 vesicles expressing the Flag-tagged ORAI proteins V102C ORAI1 and LSK/V102C ORAI1, following Cu2+-phenanthroline crosslinking in the absence or presence of STIM1CT. Multiple samples were treated in parallel for each condition. Red and black arrowheads demarcate ORAI1 dimer and monomer bands, respectively. Representative of 3–5 experiments. (d) Fraction of ORAI1 in the dimer band (mean ± SEM) from gels shown in Figure 5b–c. ****, p < 0.0001 by two-tailed Student’s t-test. No change was found in disulfide-linked dimer formation of FLAG-ORAI1LSK/V102C following STIM1CT treatment (NS, p > 0.05).
Mentions: The Cu2+-phenanthroline crosslinking assay that we developed to map ORAI1 pore architecture (44) is an independent way to probe for a conformational change at the external entrance to the pore. Treatment with Cu2+-phenanthroline crosslinks pairs of cysteine side chains that are positioned suitably and is very sensitive to the local geometry of protein side chains, able to discriminate, for example, between the ‘a’ and ‘d’ positions in the hydrophobic core of a coiled coil (45). We focused on V102, whose side chain projects into the pore one helical turn below E106. The valine > cysteine substitution needed for the crosslinking assay is conservative, replacing the valine side chain with a slightly smaller side chain of comparable hydrophobicity, and we verified that both the initial Tb3+ luminescence signal of the V102C channel and the conformational change reported by a STIM-induced increase in Tb3+ luminescence resemble those of the wildtype channel (Figure 5a).

Bottom Line: We show that interaction of STIM1 with the cytoplasmic face of the human ORAI1 channel elicits a conformational change near the external entrance to the pore, detectable at the pore Ca(2+)-binding residue E106 and the adjacent pore-lining residue V102.We demonstrate that a short nonpolar segment of the pore including V102 forms a barrier to ion flux in the closed channel, implicating the STIM1-dependent movement in channel gating.Our data explain the close coupling between ORAI1 channel gating and ion selectivity, and open a new avenue to dissect the gating, modulation and inactivation of ORAI-family channels.

View Article: PubMed Central - PubMed

Affiliation: Division of Signalling and Gene Expression, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037, USA.

ABSTRACT
The ER-resident regulatory protein STIM1 triggers store-operated Ca(2+) entry by direct interaction with the plasma membrane Ca(2+) channel ORAI1. The mechanism of channel gating remains undefined. Here we establish that STIM1 gates the purified recombinant ORAI1 channel in vitro, and use Tb(3+) luminescence and, separately, disulfide crosslinking to probe movements of the pore-lining helices. We show that interaction of STIM1 with the cytoplasmic face of the human ORAI1 channel elicits a conformational change near the external entrance to the pore, detectable at the pore Ca(2+)-binding residue E106 and the adjacent pore-lining residue V102. We demonstrate that a short nonpolar segment of the pore including V102 forms a barrier to ion flux in the closed channel, implicating the STIM1-dependent movement in channel gating. Our data explain the close coupling between ORAI1 channel gating and ion selectivity, and open a new avenue to dissect the gating, modulation and inactivation of ORAI-family channels.

Show MeSH
Related in: MedlinePlus