期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 106, 期 18, 页码 7391-7396出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0900293106
关键词
calcium signaling; DT40 cells; CRAC channel
资金
- National Institutes of Health [AI058173]
- American Heart Association
- Grants-in-Aid for Scientific Research [20249015] Funding Source: KAKEN
STIM proteins are sensors of endoplasmic reticulum (ER) luminal Ca2+ changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca2+ entry through the Orai family of highly Ca2+-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 Ca2+ release-activated Ca2+ (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.
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