National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
University of Chinese Academy of Science, Beijing 100049, China.
Sci Rep. 2016 Jul 4;6:29304. doi: 10.1038/srep29304.
2-Aminoethoxydiphenyl borate (2-APB) elicits potentiation current (Ip) on Ca(2+) release-activated Ca(2+) (CRAC) channels. An accurate investigation into this modulation mechanism would reveal how STIM1-dependent channel gating is enhanced, and benefit the future immune enhancer development. Here, we directly probed the pore diameter of CRAC channels and found that 2-APB enlarged the pore size of STIM1-activated Orai1 from 3.8 to 4.6 Å. We demonstrated that ions with small sizes, i.e., Ca(2+) and Na(+), mediated prominent 2-APB-induced Ip on the wildtype (WT) Orai1 channels of narrow pore sizes, while conducted decreased or no Ip on Orai1-V102C/A/G mutant channels with enlarged pore diameters. On the contrary, large Cs(+) ions blocked the WT channels, while displayed large 2-APB induced Ip on pore-enlarged Orai1-V102C/A/G mutant channels, and the potentiation ratio was highest on Orai1-V102C with an intermediate pore size. Furthermore, we showed that 2-APB potentiated Cs(+) current on constitutively active Orai1-V102C/A/G mutants independent of STIM1. Our data suggest that 2-APB directly dilates the pore of open Orai1 channels, both ion size and pore diameter jointly determine the amplitude of Ip on CRAC channels, and the generation of Ip requires the open state of Orai1, not STIM1 itself.
2-氨基乙氧基二苯硼酸盐（2-APB）在 Ca(2+) 释放激活的 Ca(2+)（CRAC）通道上引发增效电流（Ip）。对这种调节机制的准确研究将揭示 STIM1 依赖性通道门控如何增强，并有益于未来免疫增强剂的开发。在这里，我们直接探测了 CRAC 通道的孔径，发现 2-APB 将 STIM1 激活的 Orai1 的孔径从 3.8 增大到 4.6Å。我们证明了小尺寸的离子，即 Ca(2+) 和 Na(+)，在野生型（WT）Orai1 通道的小孔径上介导了明显的 2-APB 诱导的 Ip，而在孔径增大的 Orai1-V102C/A/G 突变体通道上则表现出减少或没有 Ip。相反，大 Cs(+) 离子阻断 WT 通道，但在孔径增大的 Orai1-V102C/A/G 突变体通道上显示出大的 2-APB 诱导的 Ip，并且在具有中间孔径的 Orai1-V102C 上具有最高的增效比。此外，我们表明 2-APB 独立于 STIM1 增强了组成激活的 Orai1-V102C/A/G 突变体上的 Cs(+) 电流。我们的数据表明，2-APB 直接扩张开放的 Orai1 通道的孔，离子大小和孔径共同决定 CRAC 通道上 Ip 的幅度，并且 Ip 的产生需要 Orai1 的开放状态，而不是 STIM1 本身。
