Functional Reconstitution and Structural Flexibility of the CRAC Channel Orai

Abstract

Calcium (Ca2+) release-activated Ca2+ (CRAC) channels mediate Ca2+ influx across the plasma membrane in response to Ca2+ store depletion in the endoplasmic reticulum (ER). CRAC channel function requires two components: the plasma membrane Ca2+ channel Orai and its regulator located in the ER membrane, the Ca2+ store sensor STIM. Using liposomes reconstituted with a purified fusion protein of human Orai1 and cytosolic fragments of human STIM1 (hO1-SS), we show that Orai1 and STIM1 are sufficient to form active CRAC channels in vitro. Reconstituted hO1-SS recapitulates CRAC channel properties as shown by detection of sodium (Na+) flux in the absence of Ca2+ and by direct detection of Ca2+ flux. 2-APB, a known CRAC channel inhibitor, blocks both fluxes. Our findings confirm that human STIM1 gates the pore of Orai1 and demonstrates that the two proteins are sufficient to form functional channels in the absence of other cellular factors. Previously, we published the crystal structure of drosophila Orai in a closed state. Here we present low-resolution X-ray diffraction data of human Orai1, which indicate an overall structure that is indistinguishable from drosophila Orai. In addition, a new 4.25 A resolution X-ray structure of drosophila Orai reveals an extended conformation of the fourth transmembrane helix (M4) at the periphery of the channel that extends into the cytosol and is strikingly different from the arrangement of these helices in the previous structure. The comparison of structures reveals conformational flexibility that starts from the M4 helices and continues into the cytosolic M4 extension helices. In all of the structures, the pore adopts the same conformation and remains closed. The conformational flexibility observed for the M4 and M4 extension helices may have a role in the binding of STIM and in signal transduction from the ER.