Stromal interaction molecule 1 (STIM1) is a widely expressed protein that functions as the endoplasmic reticulum (ER) Ca²⁺ sensor and activator of Orai1 channels. In resting cells with replete Ca²⁺ stores, an inhibitory clamp formed by the coiled-coil 1 (CC1) domain interacting with the CRAC-activation domain (CAD) of STIM1 helps keep STIM1 in a quiescent state. Following depletion of ER Ca²⁺ stores, the brake is released, allowing CAD to extend away from the ER membrane and enabling it to activate Orai1 channels. However, the molecular determinants of CC1–CAD interactions that enforce the inhibitory clamp are incompletely understood. Here, we performed Ala mutagenesis in conjunction with live-cell FRET analysis to examine residues in CC1 and CAD that regulate the inhibitory clamp. Our results indicate that in addition to previously identified hotspots in CC1⍺1 and CC3, several hydrophobic residues in CC2 and the apex region of CAD are critical for CC1–CAD interactions. Mutations in these residues loosen the CC1-CAD inhibitory clamp to release CAD from CC1 in cells with replete Ca²⁺ stores. By contrast, altering the hydrophobic residues L265 and L273 strengthens the clamp to prevent STIM1 activation. Inclusion of the inactivation domain of STIM1 helps stabilize CC1–CAD interaction in several mutants to prevent spontaneous STIM1 activation. In addition, R426C, a human disease–linked mutation in CC3, affects the clamp but also impairs Orai1 binding to inhibit CRAC channel activation. These results identify the CC2, apex, and inactivation domain regions of STIM1 as important determinants of STIM1 activation.

基质相互作用分子 1 (STIM1) 是一种广泛表达的蛋白质，充当内质网 (ER) Ca ²⁺传感器和 Orai1 通道激活剂。在具有充足 Ca ²⁺储备的静息细胞中，由卷曲螺旋 1 (CC1) 结构域与 STIM1 的 CRAC 激活结构域 (CAD) 相互作用形成的抑制钳有助于使 STIM1 保持静止状态。 ER Ca ²⁺储备耗尽后，制动器被释放，使 CAD 远离 ER 膜并使其能够激活 Orai1 通道。然而，强制抑制钳的 CC1-CAD 相互作用的分子决定因素尚不完全清楚。在这里，我们结合活细胞 FRET 分析进行 Ala 诱变，以检查 CC1 和 CAD 中调节抑制钳的残基。我们的结果表明，除了先前确定的 CC1⍺1 和 CC3 中的热点之外，CC2 和 CAD 顶端区域中的几个疏水残基对于 CC1-CAD 相互作用也至关重要。这些残基的突变会松开 CC1-CAD 抑制钳，从而在 Ca ²⁺储备充足的细胞中从 CC1 中释放 CAD。相比之下，改变疏水性残基 L265 和 L273 可以增强钳位，从而防止 STIM1 激活。包含 STIM1 失活结构域有助于稳定多个突变体中的 CC1-CAD 相互作用，以防止 STIM1 自发激活。此外，R426C（一种与人类疾病相关的 CC3 突变）会影响钳位，但也会损害 Orai1 结合，从而抑制 CRAC 通道激活。这些结果表明 STIM1 的 CC2、顶端和失活结构域区域是 STIM1 激活的重要决定因素。