From the Departments of Cellular and Molecular Physiology and.
the Secretory Physiology Section, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, and.
J Biol Chem. 2019 Apr 19;294(16):6318-6332. doi: 10.1074/jbc.RA118.006801. Epub 2019 Mar 1.
Store-operated Ca entry (SOCE) is a ubiquitous pathway for Ca influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1, STIM2, and STIM1/2 knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.
钙库操纵性钙内流（SOCE）是质膜（PM）中钙内流的普遍途径。SOCE 由内质网（ER）相关钙感应蛋白基质相互作用分子 1（STIM1）和 STIM2 介导，当 ER 钙库耗竭时，它们转变为活性构象，从而与 PM 相关的 ORAI1 通道相互作用并门控。尽管结构上同源，但 STIM1 和 STIM2 在响应不同强度的激动剂刺激时产生不同的钙特征。这些钙特征的生理功能，特别是在天然条件下，仍然不清楚。为了研究在天然条件下区分 STIM1 和 STIM2 激活 ORAI1 通道的结构特性，我们使用 CRISPR/Cas9 在 HEK293 和结直肠 HCT116 细胞中生成了 STIM1、STIM2 和 STIM1/2 敲除体。我们表明，根据细胞类型的不同，STIM2 可以在最大的储存耗尽时显著维持 SOCE。利用 SOCE 调节剂 2-氨基乙氧基二苯硼酸盐（2-APB），我们证明 2-APB 激活的储存非依赖性钙内流仅由内源性 STIM2 介导。使用分别稳定或破坏 STIM C 末端分子内相互作用的变体，我们表明 STIM2 C 末端的增加的灵活性有助于其被 2-APB 选择性地非储存依赖性激活。然而，具有增强的 C 末端灵活性的 STIM1 变体未能支持其储存非依赖性激活。STIM1/STIM2 嵌合构建体表明，N 端敏感性和 C 端灵活性之间的协调是特异性储存非依赖性 STIM2 激活所必需的。我们的结果阐明了激活特定 STIM 同工型的结构决定因素，这些见解对于同工型选择性药物靶向可能是有用的。
