Instituto de Biología y Genética Molecular (IBGM), Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), Ramón y Cajal 7, E-47005 Valladolid, Spain.
Cell Calcium. 2013 Jul;54(1):37-45. doi: 10.1016/j.ceca.2013.04.001. Epub 2013 May 2.
We use here a new very low-Ca(2+)-affinity targeted aequorin to measure the [Ca(2+)] in the endoplasmic reticulum ([Ca(2+)]ER). The new aequorin chimera has the right Ca(2+)-affinity to make long-lasting measurements of [Ca(2+)]ER in the millimolar range. Moreover, previous Ca(2+)-depletion of the ER is no longer required. The steady-state [Ca(2+)]ER obtained is 1-2 mM, higher than previously reported. In addition, we find evidence that there is significant heterogeneity in [Ca(2+)]ER among different regions of the ER. About half of the ER had a [Ca(2+)]ER of 1 mM or below, and the rest had [Ca(2+)]ER values above 1mM and in some parts even above 2 mM. About 5% of the ER was also found to have high [Ca(2+)]ER levels but to be thapsigargin-insensitive and inositol trisphosphate insensitive. The rate of refilling with Ca(2+) of the ER was almost linearly dependent on the extracellular [Ca(2+)] between 0.1 and 3 mM, and was only partially affected by mitochondrial membrane depolarization. Instead, it was significantly reduced by loading cells with chelators, and the fast chelator BAPTA was much more effective than the slow chelator EGTA. This suggests that local [Ca(2+)] microdomains connecting the store operated Ca(2+) channels with the ER Ca(2+) pumps may be important during refilling.
我们在这里使用一种新的、对钙离子亲和力非常低的靶向水母发光蛋白来测量内质网中的钙离子浓度([Ca(2+)]ER)。这种新的水母发光蛋白嵌合体具有正确的钙离子亲和力，可以在毫摩尔范围内进行长时间的内质网钙离子浓度测量。此外，先前对内质网钙离子的耗竭不再是必需的。得到的稳态内质网钙离子浓度为 1-2mM，高于之前的报道。此外，我们发现有证据表明内质网中不同区域的内质网钙离子浓度存在显著的异质性。大约一半的内质网钙离子浓度为 1mM 或以下，其余部分的内质网钙离子浓度在 1mM 以上，有些部位甚至在 2mM 以上。大约 5%的内质网也被发现具有高内质网钙离子浓度，但对 thapsigargin 不敏感，对肌醇三磷酸也不敏感。内质网的钙离子再填充率与细胞外钙离子浓度在 0.1 到 3mM 之间几乎呈线性相关，并且仅部分受到线粒体膜去极化的影响。相反，用螯合剂加载细胞会显著降低再填充率，而快速螯合剂 BAPTA 比缓慢螯合剂 EGTA 更有效。这表明在再填充过程中，连接储存操纵型钙离子通道和内质网钙离子泵的局部钙离子微区可能很重要。
