Development of CFP-based calcium probes that emit blue fluorescence

钙离子作为肌肉收缩、神经元活动和激素分泌的细胞内介质发挥着重要作用。因此，测定钙离子对细胞功能的研究具有重要意义。本研究的目的是开发基于绿色荧光蛋白(GFP)的新型蓝色荧光钙传感器。这种类型的传感器是遗传编码的，因此，它可以很容易地在特定的细胞或组织中使用特定的启动子表达。这种传感器可能成为体内钙测量的有用工具。为了制造这种传感器，我们将青色版本的GFP融合到钙调蛋白和肌球蛋白轻链激酶的M13片段中。当DNA~VAS表达HEK细胞或PC12细胞时。我们可以用CCD摄像机或激光共聚焦显微镜监测细胞内的荧光变化。有趣的是，在静止状态下，荧光更亮，而当细胞被激活时，荧光变得更暗。钙传感器中有4个钙结合部位。为了减少钙缓冲效应和改变传感器的钙敏感性，我们接下来在这三个钙结合位点引入突变。当我们破坏3个以上的钙结合部位时，传感器受到严重影响，不再用作钙传感器。另一方面，突变一个或两个钙结合位点是可以容忍的，这些突变仍然是有用的钙传感器。令人惊讶的是，尽管它们的钙敏感性受到突变的影响，但影响并不像我们预期的那么大。这些在钙结合位点上有突变的传感器可能是很好的工具，因为它们减少了钙的缓冲作用。

Calcium ions play important roles as an intracellular mediator of muscle contraction, neuronal activity and hormone secretion. Therefore, it is useful to measure calcium ion to study cellular function. The purpose of this research is to develop new Green fluorescent protein (GFP) based calcium sensors that emit blue fluorescence. This type of sensors are genetically-encoded, therefore, it can be easily expressed in specific cells or tissues using a specific promoter. This sensor may become a useful tool for in vivo calcium measurements. To make such kind of sensors we fused a cyan version of GFP to calmodulin and the M13 fragment from myosin light chain kinase. When the DNA ~vas expressed HEK cells or PC12 cells. we could monitor fluorescence changes in the cells with a CCD camera or a laser confocal microscope. Interestingly, the fluorescence was brighter at the resting condition and it became dimmer when the cells were activated. There are 4 calcium binding sites in a calcium sensor. To reduce the calcium buffering effect and to change the calcium sensitivity of the sensor, we next introduced mutations in there calcium binding sites. When we destroyed more than 3 calcium binding sites, the sensor was severely affected and it was not useful as a calcium sensor any more. On the other hand, mutating one or two calcium binding sites is tolerable and those mutants were still useful as calcium sensors. To be surprised, although their calcium sensitivities were affected by the mutations, the effects were not as large as we expected. These sensors which have mutations in calcium binding sites may be good tools because they have reduced calcium buffering effects.