TY - JOUR
T1 - STIM protein coupling in the activation of Orai channels
AU - Wang, Youjun
AU - Deng, Xiaoxiang
AU - Zhou, Yandong
AU - Hendron, Eunan
AU - Mancarella, Salvatore
AU - Ritchie, Michael F.
AU - Tang, Xiang D.
AU - Baba, Yoshihiro
AU - Kurosaki, Tomohiro
AU - Mori, Yasuo
AU - Soboloff, Jonathan
AU - Gill, Donald L.
PY - 2009/5/5
Y1 - 2009/5/5
N2 - 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-aminoethoxy- diphenyl 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 Orail 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.
AB - 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-aminoethoxy- diphenyl 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 Orail 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.
KW - Calcium signaling
KW - Crac channel
KW - Dt40 cells
UR - http://www.scopus.com/inward/record.url?scp=66149124488&partnerID=8YFLogxK
U2 - 10.1073/pnas.0900293106
DO - 10.1073/pnas.0900293106
M3 - Article
SN - 0027-8424
VL - 106
SP - 7391
EP - 7396
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 18
ER -