The earliest intracellular signals that occur after T cell activation are local, subsecond Ca²⁺ microdomains. Here, we identified a Ca²⁺ entry component involved in Ca²⁺ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca²⁺ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca²⁺ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca²⁺ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca²⁺ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca²⁺ release through RYR1, in coordination with Ca²⁺ entry through ORAI1 and STIM, rapidly increases the number of Ca²⁺ microdomains, thereby initiating spread of Ca²⁺ signals deeper into the cytoplasm to promote full T cell activation.

T细胞活化后发生的最早的细胞内信号是局部亚秒Ca ²⁺微区。在这里，我们确定了在未刺激和刺激的T细胞中参与Ca ²⁺微域形成的Ca ²⁺进入组分。在未刺激的T细胞中，自发产生小Ca ²⁺微域需要ORAI1，STIM1和STIM2。未受刺激的T细胞的超高分辨率显微镜检查发现了一个直径约300 nm的圆形亚浆膜下膜区域，并形成了预先定位的ORAI1，ryanodine受体（RYRs）和STIM1的共贴片。通过共免疫沉淀和福斯特共振能量转移研究证实了未刺激细胞中STIM1和ORAI1的预先形成的复合物。此外，在T细胞受体（TCR）刺激后的第一秒内，浆膜下间隙中Ca ²⁺微结构域的数量增加，这种作用需要ORAI1，STIM2，RYR1和Ca ²⁺动员第二信使NAADP（烟酸）腺嘌呤二核苷酸磷酸）。这些结果表明，预先形成的STIM和ORAI1簇使局部Ca ²⁺未刺激细胞中的进入事件。在TCR激活后，NAADP引起的RYR1释放的Ca ²⁺与通过ORAI1和STIM进入的Ca ²⁺协同作用，迅速增加了Ca ²⁺微结构域的数量，从而启动了Ca ²⁺信号更深地扩散到细胞质中以促进完整的T细胞活化。