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

The ER-resident regulatory protein STIM1 triggers store-operated Ca²⁺ entry by direct interaction with the plasma membrane Ca²⁺ channel ORAI1. The mechanism of channel gating remains undefined. Here we establish that STIM1 gates the purified recombinant ORAI1 channel in vitro, and use Tb³⁺ 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 Ca²⁺-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.