The C- and N-terminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels
The C- and N-terminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels
复制标题
DOI：
10.1113/jphysiol.2012.250456
复制
发表时间：
2013-06-01
影响因子：
5.5
通讯作者：
Prakriya, Murali
中科院分区：
文献类型：
--
作者：
McNally, Beth A.;Somasundaram, Agila;Prakriya, Murali
关键点中心点内质网蛋白质，基质相互作用分子1（STIM 1），通过直接与C-和N-末端的每个Orai 1亚基相互作用来激活Orai 1通道。目前关于这些位点的作用的模型植根于模块化的概念，C-末端位点被认为介导STIM 1结合，N-末端位点被认为调节通道门控。中心点在这里，我们报告说，这两个位点的功能不是那么不同：N-末端位点有助于稳定的联系STIM 1到Orai 1，相反，C-末端位点调节通道激活。除了通道激活，STIM 1结合还调节Orai 1通道离子选择性。调节离子选择性的结构要求与门控密切匹配，表明门控和渗透在Orai 1通道中紧密耦合。这些结果有助于我们理解STIM 1介导的Orai 1通道激活和通道离子选择性调节的分子要求。摘要Ca 2+释放激活的Ca 2+（CRAC）通道通过一种机制被激活，其中细胞内钙储存的耗尽导致基质相互作用分子1（STIM 1）、内质网（ER）Ca 2+传感器和Orai 1（CRAC通道蛋白）聚集在ER和细胞膜（PMs）的重叠位点。CRAC通道的重新分布是通过直接的STIM 1-Orai 1结合驱动的，这是一个重要的事件，不仅控制门控，而且调节Orai 1离子选择性。Orai 1具有两个STIM 1结合位点，分别位于细胞内的C-和N-末端。先前的研究已经提出了这些位点的模块化功能，C-末端位点被认为调节STIM 1-Orai 1结合和在ER-PM连接处捕获Orai 1，而N-末端位点介导门控。然而，在这里，我们发现在N-末端位点的各种突变损害Orai 1与STIM 1和可溶性CRAC激活结构域（CAD）的结合。通过将最小的STIM 1激活结构域（S）直接拴系到Orai 1（Orai 1-SS通道），可以在几个N-和C-末端点突变体中恢复门控，这表明在这些突变体中全长STIM 1导致的门控丧失是由于配体结合不足。相比之下，门控不能恢复突变Orai 1-SS通道携带更剧烈的删除，删除了STIM 1结合位点（1-85，73-85，或272-279 Orai 1），这表明STIM 1结合到两个网站是必不可少的通道激活。此外，离子选择性的分析表明，门控和调制的离子选择性的分子要求是相似的，但实质上不同于Orai 1斑点形成，这表明离子选择性和门控在CRAC通道中机械耦合。我们的研究结果表明，C-和N-末端的STIM 1结合位点都是必不可少的Orai 1功能的多个方面，包括STIM 1-Orai 1协会，Orai 1捕获，通道激活。
Key points center dot The endoplasmic reticulum protein, stromal interaction molecule 1 (STIM1), activates Orai1 channels by directly interacting with each Orai1 subunit at the C- and N-termini. Current models about the roles of these sites are rooted in notions of modularity, with the C-terminal site thought to mediate STIM1 binding and the N-terminal site thought to regulate channel gating. center dot Here we report that the functions of the two sites are not so distinct: the N-terminal site contributes to the stable association of STIM1 to Orai1, and, conversely, the C-terminal site regulates channel activation. center dot In addition to channel activation, STIM1 binding also modulates Orai1 channel ion selectivity. The structural requirements for modulation of ion selectivity closely match those seen for gating, suggesting that gating and permeation are closely coupled in Orai1 channels. center dot These results help us understand the molecular requirements of STIM1-mediated activation of Orai1 channels and regulation of channel ion selectivity. Abstract Ca2+ release-activated Ca2+ (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca2+ sensor, and Orai1, the CRAC channel protein, at overlapping sites in the ER and plasma membranes (PMs). The redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, an important event that not only controls gating, but also regulates Orai1 ion selectivity. Orai1 harbours two STIM1 binding sites, one each on the intracellular C- and N-termini. Previous studies have proposed modular functions for these sites, with the C-terminal site thought to regulate STIM1-Orai1 binding and trapping of Orai1 at the ER-PM junctions, and the N-terminal site mediating gating. However, here we find that a variety of mutations in the N-terminal site impair the binding of Orai1 to STIM1 and to the soluble CRAC activation domain (CAD). Gating could be restored in several N- and C-terminal point mutants by directly tethering the minimal STIM1 activation domain (S) to Orai1 (Orai1-SS channels), indicating that loss of gating in these mutants by full-length STIM1 results from insufficient ligand binding. By contrast, gating could not be restored in mutant Orai1-SS channels carrying more drastic deletions that removed the STIM1 binding sites (1-85, 73-85, or 272-279 Orai1), suggesting that STIM1 binding to both sites is essential for channel activation. Moreover, analysis of ion selectivity indicated that the molecular requirements for gating and modulation of ion selectivity are similar, yet substantively different from those for Orai1 puncta formation, suggesting that ion selectivity and gating are mechanistically coupled in CRAC channels. Our results indicate that the C- and N-terminal STIM1 binding sites are both essential for multiple aspects of Orai1 function including STIM1-Orai1 association, Orai1 trapping, and channel activation.