Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states
Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states
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DOI：
10.1073/pnas.1114814108
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发表时间：
2011-10-25
影响因子：
11.1
通讯作者：
Parker, Ian
中科院分区：
文献类型：
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作者：
Demuro, Angelo;Penna, Aubin;Parker, Ian
我们应用单分子光漂白来研究用eGFP标记并在哺乳动物细胞中表达的人Orai 1和Orai 3通道的化学计量。Orai 1在静息条件下主要以二聚体形式检测，而在与C-STIM 1共表达以激活Ca（2+）内流时主要以四聚体形式检测。Orai 1也被发现是四聚体时，与STIM 1共表达，并在固定后进行评估。我们发现，固定迅速导致Ca（2+）释放，STIM 1重新分布到质膜，STIM 1/Orai 1斑点形成，并可能导致通道处于激活状态。与这种可能性相一致的是，Orai 1在静息条件下与STIM 1在活细胞中共表达时主要以二聚体形式存在。我们进一步表明，Orai 3，像Orai 1，是二聚体在静息条件下，主要是四聚体时，激活C-STIM 1。有趣的是，在应用2-氨基乙基二苯基硼酸酯（2-APB）之前和期间发现二聚Orai 3化学计量，以在其STIM 1独立模式中激活非选择性阳离子电导。我们的结论是，人Orai 1和Orai 3通道在钙库激活过程中经历二聚体到四聚体的转变，形成一个Ca（2+）选择性孔，Orai 3在2-APB激活后形成二聚体非选择性阳离子孔。
We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in mammalian cells. Orai1 was detected predominantly as dimers under resting conditions and as tetramers when coexpressed with C-STIM1 to activate Ca(2+) influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca(2+), redistribution of STIM1 to the plasma membrane, and STIM1/Orai1 puncta formation, and may cause the channel to be in the activated state. Consistent with this possibility, Orai1 was found predominantly as a dimer when coexpressed with STIM1 in living cells under resting conditions. We further show that Orai3, like Orai1, is dimeric under resting conditions and is predominantly tetrameric when activated by C-STIM1. Interestingly, a dimeric Orai3 stoichiometry was found both before and during application of 2-aminoethyldiphenyl borate (2-APB) to activate a nonselective cation conductance in its STIM1-independent mode. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca(2+)-selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.