STIM1 gates the store-operated calcium channel ORAI1 in vitro.
Store-operated Ca(2+) entry through the plasma membrane Ca(2+) release-activated Ca(2+) (CRAC) channel in mammalian T cells and mast cells depends on the sensor protein stromal interaction molecule 1 (STIM1) and the channel subunit ORAI1. To study STIM1-ORAI1 signaling in vitro, we have expressed human ORAI1 in a sec6-4 strain of the yeast Saccharomyces cerevisiae and isolated sealed membrane vesicles carrying ORAI1 from the Golgi compartment to the plasma membrane. We show by in vitro Ca(2+) flux assays that bacterially expressed recombinant STIM1 opens wild-type ORAI1 channels but not channels assembled from the ORAI1 pore mutant E106Q or the ORAI1 severe combined immunodeficiency (SCID) mutant R91W. These experiments show that the STIM1-ORAI1 interaction is sufficient to gate recombinant human ORAI1 channels in the absence of other proteins of the human ORAI1 channel complex, and they set the stage for further biochemical and biophysical dissection of ORAI1 channel gating.
Zhou
Yubin
Y
Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital, Boston, Massachusetts, USA.
Meraner
Paul
P
Kwon
Hyoung T
HT
Machnes
Danya
D
Oh-hora
Masatsugu
M
Zimmer
Jochen
J
Huang
Yun
Y
Stura
Antonio
A
Rao
Anjana
A
Hogan
Patrick G
PG
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000378:metabolism*; D000071740:ORAI1 Protein; D011994:Recombinant Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D022142:Secretory Vesicles / Q000378:metabolism; D000071737:Stromal Interaction Molecule 1
D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D011994:Recombinant Proteins; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium