Genetically encoded bright Ca2+ probe applicable for dynamic Ca2+ imaging of dendritic spines
Genetically encoded bright Ca2+ probe applicable for dynamic Ca2+ imaging of dendritic spines
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DOI：
10.1021/ac0506837
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发表时间：
2005-09-15
影响因子：
7.4
通讯作者：
Nakai, J
中科院分区：
文献类型：
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作者：
Ohkura, M;Matsuzaki, M;Nakai, J
G-CaMP是一种基于单一绿色荧光蛋白（GFP）的Ca2+探针。G-CaMP在Ca2+结合后荧光明显增强，但其荧光微弱且pH敏感，与其他基于gfp的单探针相似。在这里，我们报告了一种改进的G-CaMP，命名为G-CaMP1.6，它可以更容易地检测细胞内Ca2+信号。G-CaMP1.6的荧光强度是G-CaMP的40倍，主要是由于量子产率的提高。此外，与G-CaMP相比，G-CaMP1.6不仅具有较低的pH敏感性，而且对离子半径与Ca2+相似的二价阳离子具有较高的选择性。与G-CaMP相比，G-CaMP 1.6 （K-d = 146 nM， Hill系数= 3.8,F-max/F-min = 4.9）的Ca2+敏感性略向高亲和力方向偏移。当在哺乳动物细胞中表达时，GCaMP1.6随着药物的作用显示出较大的荧光变化。值得注意的是，G-CaMP1.6成功地观察到神经元树突棘等微小结构中的局部Ca2+变化，这是第一次使用基于gfp的探针观察到。G-CaMP1.6 Ca2+结合位点的其他突变改变了对Ca2+的亲和力，降低了Ca2+缓冲作用。G-CaMP1.6-CaM（E140K）在Ca2+结合位点发生突变，是一种改进的探针，其亮度增加，Ca2+缓冲能力降低。
G-CaMP is a Ca2+ probe based on a single green fluorescent protein (GFP). G-CaMP shows a large fluorescence increase upon Ca2+ binding, but its fluorescence is dim and pH sensitive, similar to other single GFP-based probes. Here we report an improved G-CaMP, named G-CaMP1.6, which enables easier detection of intracellular Ca2+ signals. G-CaMP1.6 was similar to 40 times more fluorescent than G-CaMP, mainly due to an increase in quantum yield. Furthermore, compared with G-CaMP, G-CaMP1.6 had not only a lower pH sensitivity but also a higher selectivity for divalent cations having an ionic radius similar to Ca2+. Ca2+ sensitivity of G-CaMP 1.6 (K-d = 146 nM, Hill coefficient = 3.8, F-max/F-min = 4.9) was slightly shifted toward higher affinity compared with that of G-CaMP. When expressed in mammalian cells, GCaMP1.6 showed large fluorescence changes with drug applications. Notably, local Ca2+ changes in such tiny structures as dendritic spines of neurons were successfully observed with G-CaMP1.6, this being the first observation using a GFP-based probe. Additional mutations in Ca2+-binding sites of G-CaMP1.6 shifted the affinity for Ca2+ and reduced the Ca2+-buffering effect. G-CaMP1.6-CaM(E140K), which has a mutation in the Ca2+ binding site, is an improved probe with its increased brightness and reduced Ca2+-buffering capacity.