Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+
Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+
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DOI：
10.1016/j.ceca.2003.10.005
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发表时间：
2004-05-01
期刊：
影响因子：
4
通讯作者：
Persechini, A
中科院分区：
文献类型：
--
作者：
Black, DJ;Tran, QK;Persechini, A
我们描述了一种新的基因编码的荧光生物传感器的设计，表征和应用，用于细胞内检测游离Ca 2 +-钙调素和apocalmodulin，这两者一起包括可用的钙调素浓度。该生物传感器结合两种形式的钙调素的表观Kd值为3 μ M，并具有动力学特性，使其适合于监测动态变化的亚秒级的时间尺度。它可以与荧光Ca 2+指示剂indo-1结合使用，允许同时监测可用的钙调蛋白和游离Ca 2+浓度。我们已经确定了一个细胞内可用的钙调素浓度为8.8 +/- 2.2 μ M的静息条件下，在人肾细胞系稳定表达的生物传感器。通过激动剂、钙库操作的钙进入或离子载体升高细胞内游离钙浓度导致可用钙调素的钙依赖性消耗。可用钙调素浓度与游离Ca 2+浓度的归一化值图拟合消耗曲线，协同系数为1.8，[Ca 2 +]（50）为850 nM。在游离Ca 2+浓度高于4 μ M时，没有可检测到的钙调蛋白与生物传感器的结合，这与在这些条件下可用的钙调蛋白浓度小于或等于200 nM以及钙调蛋白结合位点的总体过量一致。(C)2003 Elsevier Ltd.保留所有权利。
We describe the design, characterization and application of a new genetically encoded fluorescent biosensor for intracellular detection of both free Ca2+-calmodulin and apocalmodulin, which together comprise the available calmodulin concentration. The biosensor binds both forms of calmodulin with an apparent K-d value of 3 muM, and has kinetic properties making it suitable for monitoring dynamic changes on a subsecond time scale. It can be used in conjunction with the fluorescent Ca2+-indicator, indo-1, allowing the available calmodulin and free Ca2+ concentrations to be monitored concurrently. We have determined an intracellular available calmodulin concentration of 8.8 +/- 2.2 muM under resting conditions in a human kidney cell line stably expressing the biosensor. Elevation of the intracellular free Ca2+ concentration by agonist, store-operated Ca2+-entry or ionophore results in Ca2+-dependent consumption of the available calmodulin. A plot of normalized values for the available calmodulin concentration versus the free Ca2+ concentration fits a consumption curve with a cooperativity coefficient of 1.8 and a [Ca2+](50) of 850 nM. There is no detectible binding of calmodulin to the biosensor above a free Ca2+ concentration of similar to4 muM, consistent with an available calmodulin concentration less than or equal to200 nM under these conditions, and an overall excess of calmodulin-binding sites. (C) 2003 Elsevier Ltd. All rights reserved.