Osibow Karin, Malli Roland, Kostner Gerhard M, Graier Wolfgang F
Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, 8010 Graz, Austria.
J Biol Chem. 2006 Feb 24;281(8):5017-5025. doi: 10.1074/jbc.M508583200. Epub 2005 Dec 20.
Genetically encoded Ca2+ indicators are outstanding tools for the assessment of intracellular/organelle Ca2+ dynamics. Basically, most indicators contain the Ca2+-binding site of a (mutated) cytosolic protein that interacts with its natural (mutated) interaction partner upon binding of Ca2+. Consequently, a change in the structure of the sensor occurs that, in turn, alters the fluorescent properties of the sensor. Herein, we present a new type of genetically encoded Ca2+ indicator for the endoplasmic reticulum (ER) (apoK1-er (W. F. Graier, K. Osibow, R. Malli, and G. M. Kostner, patent application number 05450006.1 at the European patent office)) that is based on a single kringle domain from apolipoprotein(a), which is flanked by yellow and cyan fluorescent protein at the 3'- and 5'-ends, respectively. Notably, apoK1-er does not interact with Ca2+ itself but serves as a substrate for calreticulin, the main constitutive Ca2+-binding protein in the ER. ApoK1-er assembles with calreticulin and the protein disulfide isomerase ERp57 and undergoes a conformational shift in a Ca2+-dependent manner that allows fluorescence resonance energy transfer between the two fluorophores. This construct primarily offers three major advantages compared with the already existing probes: (i) it resolves perfectly the physiological range of the free Ca2+ concentration in the ER, (ii) expression of apoK1-er does not affect the Ca2+ buffering capacity of the ER, and (iii) apoK1-er is not inactivated by binding of constitutive interaction partners that prevent Ca2+-dependent conformational changes. These unique characteristics of apoK1-er make this sensor particularly attractive for studies on ER Ca2+ signaling and dynamics in which alteration of Ca2+ fluctuations by expression of any additional Ca2+ buffer essentially has to be avoided.
基因编码的钙离子指示剂是评估细胞内/细胞器钙离子动态变化的出色工具。基本上，大多数指示剂都包含一种（突变的）胞质蛋白的钙离子结合位点，该位点在钙离子结合后会与其天然（突变的）相互作用伙伴相互作用。因此，传感器的结构会发生变化，进而改变传感器的荧光特性。在此，我们展示了一种新型的内质网（ER）基因编码钙离子指示剂（apoK1-er（W.F.格雷尔、K.奥西博、R.马利和G.M.科斯特纳，欧洲专利局专利申请号05450006.1）），它基于载脂蛋白(a)的单个kringle结构域，其3'端和5'端分别侧翼有黄色和青色荧光蛋白。值得注意的是，apoK1-er本身不与钙离子相互作用，而是作为内质网中主要的组成型钙离子结合蛋白钙网蛋白的底物。ApoK1-er与钙网蛋白和蛋白二硫键异构酶ERp57组装在一起，并以钙离子依赖的方式发生构象变化，从而允许两个荧光团之间进行荧光共振能量转移。与现有的探针相比，这种构建体主要具有三个主要优点：（i）它能完美解析内质网中游离钙离子浓度的生理范围，（ii）apoK1-er的表达不会影响内质网的钙离子缓冲能力，（iii）apoK1-er不会因组成型相互作用伙伴的结合而失活，这些伙伴会阻止钙离子依赖的构象变化。ApoK1-er的这些独特特性使得该传感器对于内质网钙离子信号传导和动态变化的研究特别有吸引力，在这些研究中，必须避免因表达任何额外的钙离子缓冲剂而改变钙离子波动。
