Demaurex Nicolas, Frieden Maud
Department of Physiology, University of Geneva Medical Center, 1, Michel-Servet, CH-1211 4, Geneva, Switzerland.
Cell Calcium. 2003 Aug;34(2):109-19. doi: 10.1016/s0143-4160(03)00081-2.
The free ER Ca(2+) concentration, Ca(2+), is a key parameter that determines both the spatio-temporal pattern of Ca(2+) signals as well as the activity of ER-resident enzymes. Obtaining accurate, time-resolved measurements of the Ca(2+) activity within the ER is thus critical for our understanding of cell signaling. Such measurements, however, are particularly challenging given the highly dynamic nature of Ca(2+) signals, the complex architecture of the ER, and the difficulty of addressing probes specifically into the ER lumen. Prompted by these challenges, a number of ingenious approaches have been developed over the last years to measure ER Ca(2+) by optical means. The two main strategies used to date are Ca(2+)-sensitive synthetic dyes trapped into organelles and genetically encoded probes, based either on the photoprotein aequorin or on the green fluorescent protein (GFP). The GFP-based Ca(2+) indicators comprise the camgaroo and pericam probes based on a circularly permutated GFP, and the cameleon probes, which rely on the fluorescence resonance energy transfer (FRET) between two GFP mutants of different colors. Each approach offers unique advantages and suffers from specific drawbacks. In this review, we will discuss the advantages and pitfalls of using the genetically encoded "cameleon" Ca(2+) indicators for ER Ca(2+) measurements.
内质网游离钙离子浓度Ca(2+)是一个关键参数，它既决定了钙离子信号的时空模式，也决定了内质网驻留酶的活性。因此，对内质网内钙离子活性进行准确的、时间分辨的测量，对于我们理解细胞信号传导至关重要。然而，鉴于钙离子信号的高度动态性质、内质网的复杂结构以及将探针特异性地靶向内质网腔的困难，这样的测量极具挑战性。受这些挑战的推动，在过去几年中已经开发了许多巧妙的方法来通过光学手段测量内质网钙离子。迄今为止使用的两种主要策略是捕获到细胞器中的钙离子敏感合成染料和基于光蛋白水母发光蛋白或绿色荧光蛋白(GFP)的基因编码探针。基于GFP的钙离子指示剂包括基于环状排列GFP的袋鼠和钙 pericam探针，以及依赖于两种不同颜色GFP突变体之间荧光共振能量转移(FRET)的cameleon探针。每种方法都有其独特的优点和特定的缺点。在这篇综述中，我们将讨论使用基因编码的“cameleon”钙离子指示剂进行内质网钙离子测量的优点和缺陷。
