A novel Ca2+ indicator protein using FRET and calpain-sensitive linker.
A novel Ca2+ indicator protein using FRET and calpain-sensitive linker.
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DOI：
10.1016/j.bbrc.2005.08.089
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发表时间：
2005-10
影响因子：
3.1
通讯作者：
Kenji Takatsuka;T. Ishii;H. Ohmori
中科院分区：
文献类型：
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作者：
Kenji Takatsuka;T. Ishii;H. Ohmori
在这里，我们报告了一种基于fret的钙指示蛋白的性质。我们将ECFP和EYFP与α-spectrin calpain敏感序列结合，构建了具有GAP-43 n端棕榈酰化信号的串联融合蛋白F2C。此前有报道称，calpain裂解了一个类似的ECFP-EYFP融合蛋白，该融合蛋白由calpain敏感的α-spectrin （fodrin）序列连接。出乎意料的是，F2C没有被calpain切割，但在大鼠初级小脑培养的浦肯野细胞或体内被编码F2C的Sindbis病毒感染的脑干神经元中短暂感染时，显示出Ca2+指示剂的特性。随着胞内Ca2+浓度（[Ca2+]i）的升高，480nm/535nm的发射比不断增加。F2C具有Ca2+敏感性，表观解离常数（Kdfor Ca2+）为150nM，并且在同时测量[Ca2+]时表现出与Fura-2相似的动力学。这些性质的F2C是有用的Ca2+指示剂。
Here, we report the properties of a FRET-based calcium indicator protein. We constructed a tandem fusion protein, named F2C, of ECFP and EYFP combined with calpain-sensitive sequences of α-spectrin, with N-terminal palmitoylation signal of GAP-43. It was previously reported that calpain cleaved a similar ECFP–EYFP fusion protein linked by a calpain-sensitive sequence of α-spectrin (fodrin). Unexpectedly, F2C was not cleaved by calpain, but demonstrated properties of a Ca2+indicator when transiently infected in Purkinje cells of rat primary cerebellar culture or in the brainstem neurons infected in vivo using Sindbis virus encoding F2C. The emission ratio of 480nm/535nm was repeatedly increased when the intracellular Ca2+concentration ([Ca2+]i) was raised. F2C had a Ca2+sensitivity with an apparent dissociation constant (Kdfor Ca2+) of 150nM, and demonstrated kinetics that paralleled Fura-2 when [Ca2+]iwas measured simultaneously. These properties of F2C are useful to be a Ca2+indicator.