Store-operated Ca2+ entry (SOCE) is critical for numerous Ca2+-related processes. The activation of SOCE requires engagement between stromal interaction molecule 1 (STIM1) molecules on the endoplasmic reticulum and Ca2+ release-activated channel (CRAC) Orai on the plasma membrane. However, the molecular details of their interactions remain elusive. Here, we analyzed STIM1-Orai interactions using synthetic peptides derived from the N- and C-termini of Orai channels (Orai-NT and Orai-CT, respectively) and purified fragments of STIM1. The binding of STIM1 to Orai-NT is hydrophilic based, whereas binding to the Orai-CT is mostly hydrophobic. STIM1 decreases its affinity for Orai-CT when Orai-NT is present, supporting a stepwise interaction. Orai3-CT exhibits stronger binding to STIM1 than Orai1-CT, largely due to the shortness of one helical turn. The role of newly identified residues was confirmed by co-immunoprecipitation and Ca2+ imaging using full-length molecules. Our results provide important insight into CRAC gating by STIM1.

中文翻译：

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STIM1 以顺序方式与 Orai 通道的末端相互作用。

存储操作的 Ca2+ 进入 (SOCE) 对于许多 Ca2+ 相关过程至关重要。SOCE 的激活需要内质网上的基质相互作用分子 1 (STIM1) 分子与质膜上的 Ca2+ 释放激活通道 (CRAC) Orai 之间的结合。然而，它们相互作用的分子细节仍然难以捉摸。在这里，我们使用来自 Orai 通道 N 端和 C 端（分别为 Orai-NT 和 Orai-CT）的合成肽和 STIM1 的纯化片段分析了 STIM1-Orai 相互作用。STIM1 与 Orai-NT 的结合是基于亲水性的，而与 Orai-CT 的结合主要是疏水性的。当 Orai-NT 存在时，STIM1 会降低其对 Orai-CT 的亲和力，支持逐步相互作用。Orai3-CT 与 STIM1 的结合力强于 Orai1-CT，这主要是由于一圈螺旋较短。新鉴定的残基的作用通过免疫共沉淀和使用全长分子的 Ca2+ 成像得到证实。我们的结果为 STIM1 的 CRAC 门控提供了重要的见解。