Ca2+是细胞内重要的第二信使，调控细胞生长、增殖、免疫反应等多种重要的细胞活动。作为Ca2+信号的重要来源，SOCE（store-operated Ca2+ entry）由位于内质网的STIM1蛋白和细胞膜上的Orai1蛋白共同介导，其中Orai1的六聚体蛋白组成了通透Ca2+的通道。SOCE与许多疾病密切相关，STIM1或Orai1功能丧失突变体引起的SOCE功能紊乱将导致患者出现T细胞功能受损、免疫功能障碍、严重的感染等多种症状。因此，深入理解Orai1激活的分子机制以及开发相应的钙激活工具是至关重要的。目前基于Orai1的钙信号激活工具还局限于光遗传学手段，且均存在产生的钙信号较小的缺点。因此，还需要进一步深入探究Orai1激活机制，进而开发新型的、钙反应更大的钙信号工具，为钙功能紊乱引发的疾病治疗提供理论依据。

为了进一步研究Orai1的激活机制，本研究对合作者利用分子动力学模拟和简振模式分析建立的Orai“扭转-开放”的激活模型进行了实验验证。通过分子生物学手段，结合多种活细胞荧光成像技术及全细胞膜片钳等方法，对模型提出的R155-E221和K157-E245两个盐桥的关键作用进行了验证，证实它们对Orai1激活是必需的，从而为该激活假说提供了实验证据。

该模型提示Orai1的第三跨膜区在其激活过程中起到关键作用。为构建碱激活的钙信号工具，本研究针对TM3中富含极性氨基酸区域的关键氨基酸制备了饱和突变库，建立了基于SOCE通路的荧光素酶报告系统和钙成像实验的两级筛选体系，筛选获得了几个碱激活的Orai1突变体。经进一步的测试后，确定了Orai1-E173K突变体为最优的碱激活钙通道（APAC）。APAC可以直接被胞外碱性pH可逆激活，产生高度钙离子选择性的钙内流。与现有的光遗传学钙信号工具相比，APAC具有不影响内源SOCE信号，钙反应更大的优点。本论文进而制备了背景低、灵敏度高的“碱激活-靶蛋白表达”系统，实现了用胞外碱化来控制目的基因表达。因此，本研究丰富了基于Orai1蛋白的钙信号激活工具，该成果将为钙信号紊乱引起的相关疾病的治疗带来新的思路、提供新的解决手段。

Ca²⁺ is an important second messenger that modulates a wide range of cell functions including cell growth, proliferation, and immune responses. Store-operated Ca²⁺ entry (SOCE), as a critical source of Ca²⁺ influxes, is mediated by the ER-resident STIM1 protein and the pore-forming protein Orai1 on plasma membrane. Aberrant SOCE caused by loss of function mutantions in STIM1 or Orai1 may lead to T cell impairments, immune dysfunction, and severe infections in patients. Thus it is crucial to elucidate the molecular mechanisms of Orai1 activation and to develop Orai1-based Ca²⁺ actuators. Currently, nearly all specific Ca²⁺ actuators are limited to optogenetic approaches, and suffer from poor Ca²⁺ response. Therefore, it’s essential to illucidate more Orai1 activation mechanism, and develop novel Orai1-based Ca²⁺ entry tools.

To gain more information on the Orai1 activation mechanism, we tested the ‘‘twist-to-open’’ activation model established with combined NMA and MD simulations by our collaborator. The working model proposed the existence of two salt bridges within Orai1 that may be cricital for its activation. With a combination of molecular biological methods, together with live cell fluorescence imaging and whole-cell patch clamp techniques, we showed that these two salt bridges were essential for Orai1 activation, providing experimental evidence to support the “twist-to-open” hypothesis. The model suggests that TM3 of Orai1 plays a key role in the channel activation. To obtain alkali activated Ca²⁺ channels, we then carried out saturation mutations in the polar amino acid-enriched region of TM3, and identified several Orai1 mutants that could be activated by alkali with a two-step screening system based on SOCE-dependent luciferase reporter system and Ca²⁺ imaging assay. Further tests led us to select Orai1-E173K mutant as alkaline pH_o activated calcium channel (APAC). APAC can be directly and reversibly activated by extracellular alkaline pH to produce calcium influx with highly Ca²⁺-selectivity. Compared with the existing optogenetic Ca²⁺actuators, APAC showed minimal effect on endogenous SOCE, and much larger Ca²⁺ responses. In addition, we setted up an "alkaline activation - protein expression" system with low background activity and high sensitivity, showing it could be a proof-of-principle tool for alkali-controlled gene expression. Overall, our APAC tool expanded Ca²⁺ specific actuators, providing new potential strategies for the treatment of related diseases.