Leon-Aparicio Daniel, Pacheco Jonathan, Chavez-Reyes Jesus, Galindo Jose M, Valdes Jesus, Vaca Luis, Guerrero-Hernandez Agustin
Department of Biochemistry, Cinvestav, Mexico City, 07000, Mexico.
Department of Cell Biology and Development, Instituto de Fisiología Celular, UNAM, Mexico City, 04510, Mexico.
Cell Calcium. 2017 Jul;65:91-101. doi: 10.1016/j.ceca.2017.01.012. Epub 2017 Jan 23.
We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca leak using synthetic Ca indicators that report changes in both the cytoplasmic ([Ca]) and the luminal ER ([Ca]) Ca concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca] but did not decrease the ER Ca leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IPR and only Orai3 channel supported the 2-APB-induced ER Ca leak, while Orai1 and Orai2 inhibited this type of ER Ca leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca leak but did not modify the ER Ca leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca] response after TG but only when the ER store had been overloaded with Ca by eliminating the acidic internal Ca store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca leak but forms an ER Ca leak channel that is limiting the overloading with Ca of the ER store.
我们利用能报告细胞质（[Ca]）和内质网腔（[Ca]）钙浓度变化的合成钙指示剂，在HeLa细胞中研究了2-APB诱导的内质网钙泄漏的分子本质。我们检验了这样一个假说，即Orai通道参与了在配套论文中所描述的2-APB诱导的内质网钙泄漏。据报道，显性负性Orai1 E106A突变体的表达可阻断所有三种类型Orai通道的活性，它抑制了2-APB对[Ca]的作用，但在毒胡萝卜素（TG）处理后并未降低内质网钙泄漏。Orai3通道与表达的IPR共定位，而Orai1和Orai2则不共定位，并且只有Orai3通道支持2-APB诱导的内质网钙泄漏，而Orai1和Orai2抑制这种类型的内质网钙泄漏。降低Orai3的表达可抑制2-APB诱导的内质网钙泄漏，但不会改变用TG抑制SERCA泵所揭示的内质网钙泄漏。然而，降低Orai3通道的表达在TG处理后会导致更大的[Ca]反应，但只有在内质网钙库通过用巴弗洛霉素消除酸性内部钙库而被钙过载时才会出现这种情况。这些数据表明，Orai3通道不参与TG所揭示的内质网钙泄漏，而是形成一个内质网钙泄漏通道，该通道限制了内质网钙库的钙过载。
