STIM protein coupling in the activation of Orai channels
STIM protein coupling in the activation of Orai channels
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DOI：
10.1073/pnas.0900293106
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发表时间：
2009-05-05
影响因子：
11.1
通讯作者：
Gill, Donald L.
中科院分区：
文献类型：
--
作者：
Wang, Youjun;Deng, Xiaoxiang;Gill, Donald L.
STIM蛋白是内质网（ER）腔Ca 2+变化的传感器，并快速移位到质膜（PM）连接处附近以激活Ca 2+通过高Ca 2+选择性“钙库操纵”通道（SOC）的奥赖家族进入。剖析STIM-Orai耦合过程受到ER-PM结域的抽象性质的限制。为了克服这个问题，我们研究了耦合使用STIM嵌合体和细胞质C-末端结构域的STIM 1和STIM 2（S1 ct和S2 ct），并确定了一个基本的行动，强大的SOC改性剂，2-氨基乙氧基-二苯基硼酸酯（2-APB），其机制已经逃避了最近的审查。我们发现，2-APB诱导深刻的，快速的，和直接的相互作用之间的S1 ct或S2 ct和Orai 1，影响完全的Ca 2+释放激活的Ca 2+（CRAC）电流激活。短的235-505 S1 ct螺旋线圈区域足以进行功能性Orai 1偶联。添加2-APB后，YFP标记的S1 ct或S2 ct片段从胞质溶胶中清除，与Orai 1-CFP强烈结合，FRET快速增加，CRAC电流瞬时增加至基础水平以上200倍。在STIM 1/STIM 2（-/-）DT 40鸡B细胞中发生了功能性S1 ct-Orai 1偶联，表明ct片段独立于天然STIM蛋白而起作用。2-APB诱导的S1 ct-Orai 1和S2-ct-Orai 1复合物快速重组为离散的共定位PM簇，其保持稳定> 100 s，远远超过CRAC激活和随后的失活。除了定义2-APB的作用外，锁定的STIMct-Orai复合物还提供了一种潜在的有用探针来从结构上检查偶联。
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.