Identification of molecular determinants that govern distinct STIM2 activation dynamics

The endoplasmic reticulum (ER) Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2, which connect ER Ca2+ depletion with extracellular Ca2+ influx, are crucial for the maintenance of Ca2+ homeostasis in mammalian cells. Despite the recent progress in unraveling the role of STIM2 in Ca2+ signaling, the mechanistic underpinnings of its activation remain underexplored. We use an engineering approach to direct ER-resident STIMs to the plasma membrane (PM) while maintaining their correct membrane topology, as well as Förster resonance energy transfer (FRET) sensors that enabled in cellulo real-time monitoring of STIM activities. This allowed us to determine the calcium affinities of STIM1 and STIM2 both in cellulo and in situ, explaining the current discrepancies in the literature. We also identified the key structural determinants, especially the corresponding G residue in STIM1, which define the distinct activation dynamics of STIM2. The chimeric E470G mutation could switch STIM2 from a slow and weak Orai channel activator into a fast and potent one like STIM1 and vice versa. The systemic dissection of STIM2 activation by protein engineering sets the stage for the elucidation of the regulation and function of STIM2-mediated signaling in mammals.内质网（endoplasmic reticulum, ER）钙离子传感器基质相互作用分子1（stromal interaction molecule 1, STIM1）与STIM2可将内质网钙耗竭与细胞外钙内流相耦联，是维持哺乳动物细胞钙稳态的关键蛋白。尽管目前在阐明STIM2在钙信号通路中的作用方面已取得一定进展，但其激活的分子机制仍有待深入探究。本研究采用蛋白质工程策略，将内质网驻留的STIM蛋白定向引导至质膜（plasma membrane, PM），同时保留其正确的膜拓扑结构；此外还构建了可在细胞内实时监测STIM活性的福斯特共振能量转移（Förster resonance energy transfer, FRET）传感器。借此，我们得以在细胞内及原位条件下测定STIM1与STIM2的钙亲和力，从而解释了当前文献中存在的研究分歧。我们还鉴定出了决定STIM2独特激活动力学特性的关键结构位点，尤其是STIM1中对应的甘氨酸残基。嵌合型E470G突变可将STIM2从一种激活缓慢且作用微弱的Orai通道激活因子，转变为类似STIM1的快速强效激活因子，反之亦然。通过蛋白质工程对STIM2激活过程进行系统性解析，为阐明哺乳动物体内STIM2介导的信号通路的调控机制与生物学功能奠定了基础。