Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Poland.
J Neurochem. 2013 Sep;126(6):727-38. doi: 10.1111/jnc.12320. Epub 2013 Jun 12.
In non-excitatory cells, stromal interaction molecule 1 (STIM1) and STIM2 mediate store-operated calcium entry via an interaction with ORAI1 calcium channels. However, in neurons, STIM2 over-expression appears to play a role in calcium homeostasis that is different from STIM1 over-expression. The aim of this study was to establish the role and localization of native STIM2 in the neuronal cell. Co-immunoprecipitation experiments revealed that the interaction between endogenous STIM2 and ORAI1 was greater in a low-calcium medium than in a high-calcium medium. Using a Proximity Ligation Assay (PLA), the number of apparent complexes of endogenous STIM2 with ORAI1 was quantified. No change in the number of PLA signals was observed in the presence of thapsigargin, which depletes calcium from the endoplasmic reticulum (ER). However, the number of apparent STIM2-ORAI1 complexes increased when intracellular and subsequently ER calcium concentrations were decreased by BAPTA-AM or a low-calcium medium. Both Fura-2 acetoxymethyl ester calcium imaging and PLA in the same neuronal cell indicated that the calcium responses correlated strongly with the number of endogenous STIM2-ORAI1 complexes. The small drop in calcium levels in the ER caused by decreased intracellular calcium levels appeared to initiate the calcium-sensitive and thapsigargin-insensitive interaction between STIM2 and ORAI1. We show in neuronal somata the formation of endogenous complexes of stromal interaction molecule 2 (STIM2) with ORAI1 calcium channels. Their number increased when intracellular Ca²⁺ concentrations were decreased by the Ca²⁺ chelator BAPTA-AM or a low-calcium medium (EGTA), but did not in the presence of thapsigargin (TG). We conclude that the small drop of Ca²⁺ level in endoplasmic reticulum, due to the decreased level of intracellular Ca²⁺, is sufficient to trigger STIM2-ORAI1 complex formation in a thapsigargin-insensitive manner.
在非兴奋细胞中，基质相互作用分子 1（STIM1）和 STIM2 通过与 ORAI1 钙通道相互作用介导钙库操纵的钙内流。然而，在神经元中，STIM2 的过表达似乎在钙稳态中发挥作用，与 STIM1 的过表达不同。本研究旨在确定神经元细胞中天然 STIM2 的作用和定位。共免疫沉淀实验表明，低钙培养基中内源性 STIM2 与 ORAI1 的相互作用大于高钙培养基。使用邻近连接分析（PLA），量化了内源性 STIM2 与 ORAI1 的明显复合物的数量。在用 thapsigargin 处理时，没有观察到 PLA 信号数量的变化，thapsigargin 从内质网（ER）耗尽钙。然而，当通过 BAPTA-AM 或低钙培养基降低细胞内和随后的 ER 钙浓度时，明显的 STIM2-ORAI1 复合物的数量增加。同一神经元细胞中的 Fura-2 乙酰氧甲酯钙成像和 PLA 均表明钙反应与内源性 STIM2-ORAI1 复合物的数量密切相关。细胞内钙水平降低导致 ER 中钙水平的微小下降似乎引发了钙敏感和 thapsigargin 不敏感的 STIM2 与 ORAI1 之间的相互作用。我们在神经元胞体中显示了基质相互作用分子 2（STIM2）与 ORAI1 钙通道的内源性复合物的形成。当通过 Ca²⁺螯合剂 BAPTA-AM 或低钙培养基（EGTA）降低细胞内 Ca²⁺浓度时，它们的数量增加，但在 thapsigargin（TG）存在时没有增加。我们得出结论，由于细胞内 Ca²⁺水平降低，内质网中 Ca²⁺水平的微小下降足以触发以 thapsigargin 不敏感的方式形成 STIM2-ORAI1 复合物。
