Greotti Elisa, Wong Andrea, Pozzan Tullio, Pendin Diana, Pizzo Paola
Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, Padua 35121, Italy.
Neuroscience Institute-Italian National Research Council (CNR), Padua 35121, Italy.
Sensors (Basel). 2016 Sep 2;16(9):1419. doi: 10.3390/s16091419.
Calcium ion (Ca(2+)) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca(2+) store and the free Ca(2+) concentration ([Ca(2+)]) within its lumen ([Ca(2+)]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca(2+) sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca(2+) probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca(2+), low dynamic range, and an affinity for the cation that is too high for the levels of [Ca(2+)] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca(2+) affinity and dynamic range for monitoring [Ca(2+)] variations within the ER. As an example, resting [Ca(2+)]ER have been evaluated in a known paradigm of altered ER Ca(2+) homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer's Disease-linked protein Presenilin 2 (PS2). The lower Ca(2+) affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca(2+) content in cells expressing mutated PS2, compared to controls.
钙离子（Ca(2+)）是一种普遍存在的细胞内信使，其浓度变化几乎会影响细胞生命的方方面面。内质网（ER）是细胞内主要的钙离子储存库，其腔内的游离钙离子浓度（[Ca(2+)]）可达到高于1 mM的水平。在过去几年中，已经开发了几种基因编码的内质网靶向钙离子传感器。然而，它们中的大多数是非比率型的，因此，其信号在活细胞中难以校准，并且会受到焦平面移动和样品人为移动的影响。另一方面，现有的比率型钙离子探针存在不同的缺点，例如对钙离子的双解离常数（Kd）、低动态范围以及对阳离子的亲和力过高，不适用于内质网腔内的[Ca(2+)]水平。在这里，我们报告了一种最近生成的内质网靶向、基于荧光共振能量转移（FRET）的变色龙探针D4ER的特性，其特点是具有合适的钙离子亲和力和动态范围，可用于监测内质网内的[Ca(2+)]变化。例如，在已知的内质网钙离子稳态改变的范例中，即在表达家族性阿尔茨海默病相关蛋白早老素2（PS2）突变形式的细胞中，评估了静息[Ca(2+)]ER。与先前生成的D1ER相比，D4ER探针的钙离子亲和力较低，这使得与对照相比，能够检测到表达突变PS2的细胞中内质网钙离子含量明显更显著的降低。
