STIM protein coupling in the activation of Orai channels.
STIM proteins are sensors of endoplasmic reticulum (ER) luminal Ca(2+) changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca(2+) entry through the Orai family of highly Ca(2+)-selective "store-operated" channels (SOCs). Dissecting the STIM-Orai coupling process is restricted by the abstruse nature of the ER-PM junctional domain. To overcome this problem, we studied coupling by using STIM chimera and cytoplasmic C-terminal domains of STIM1 and STIM2 (S1ct and S2ct) and identifying a fundamental action of the powerful SOC modifier, 2-aminoethoxydiphenyl borate (2-APB), the mechanism of which has eluded recent scrutiny. We reveal that 2-APB induces profound, rapid, and direct interactions between S1ct or S2ct and Orai1, effecting full Ca(2+) release-activated Ca(2+) (CRAC) current activation. The short 235-505 S1ct coiled-coil region was sufficient for functional Orai1 coupling. YFP-tagged S1ct or S2ct fragments cleared from the cytosol seconds after 2-APB addition, binding avidly to Orai1-CFP with a rapid increase in FRET and transiently increasing CRAC current 200-fold above basal levels. Functional S1ct-Orai1 coupling occurred in STIM1/STIM2(-/-) DT40 chicken B cells, indicating ct fragments operate independently of native STIM proteins. The 2-APB-induced S1ct-Orai1 and S2-ct-Orai1 complexes undergo rapid reorganization into discrete colocalized PM clusters, which remain stable for >100 s, well beyond CRAC activation and subsequent deactivation. In addition to defining 2-APB's action, the locked STIMct-Orai complex provides a potentially useful probe to structurally examine coupling.
Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Youjun
Y
Wang
Xiaoxiang
X
Deng
Yandong
Y
Zhou
Eunan
E
Hendron
Salvatore
S
Mancarella
Michael F
MF
Ritchie
Xiang D
XD
Tang
Yoshihiro
Y
Baba
Tomohiro
T
Kurosaki
Yasuo
Y
Mori
Jonathan
J
Soboloff
Donald L
DL
Gill
D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D015220:Calcium Channels; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins