Purpose

In the brain, astrocytes are non-excitable cells that undergo rapid morphological changes when stimulated by the excitatory neurotransmitter glutamate. We developed a chemical probe to monitor how glutamate affects the density and distribution of astrocytic L-type voltage-gated calcium channels (LTCC).

Procedures

The imaging probe FluoBar1 was created from a barbiturate ligand modified with a fluorescent coumarin moiety. The probe selectivity was examined with colocalization analyses of confocal fluorescence imaging in U118-MG and transfected COS-7 cells. Living cells treated with 50 nM FluoBar1 were imaged in real time to reveal changes in density and distribution of astrocytic LTCCs upon exposure to glutamate.

Results

FluoBar1 was synthesized in ten steps. The selectivity of the probe was demonstrated with immunoblotting and confocal imaging of immunostained cells expressing the Ca_V1.2 isoform of LTCCs proteins. Applying FluoBar1 to astrocyte model cells U118-MG allowed us to measure a fivefold increase in fluorescence density of LTCCs upon glutamate exposure.

Conclusions

Imaging probe FluoBar1 allows the real-time monitoring of LTCCs in living cells, revealing for first time that glutamate causes a rapid increase of LTCC membranar density in astrocyte model cells. FluoBar1 may help tackle previously intractable questions about LTCC dynamics in cellular events.

中文翻译：

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用于监测星形胶质细胞谷氨酸信号中 L 型电压门控钙通道实时再分布的成像探针的设计

目的

在大脑中，星形胶质细胞是不可兴奋的细胞，当受到兴奋性神经递质谷氨酸的刺激时会发生快速的形态变化。我们开发了一种化学探针来监测谷氨酸如何影响星形细胞 L 型电压门控钙通道 (LTCC) 的密度和分布。

程序

成像探针 FluoBar1 由经荧光香豆素部分修饰的巴比妥配体制成。在 U118-MG 和转染的 COS-7 细胞中用共聚焦荧光成像的共定位分析检查探针选择性。用 50 nM FluoBar1 处理的活细胞实时成像，以揭示暴露于谷氨酸后星形胶质细胞 LTCC 的密度和分布变化。

结果

FluoBar1 分十步合成。通过对表达LTCCs 蛋白Ca _V 1.2 同种型的免疫染色细胞进行免疫印迹和共聚焦成像，证明了探针的选择性。将 FluoBar1 应用于星形胶质细胞模型细胞 U118-MG 使我们能够测量 LTCC 在谷氨酸暴露后荧光密度增加了五倍。

结论

成像探针 FluoBar1 允许实时监测活细胞中的 LTCC，首次揭示谷氨酸导致星形胶质细胞模型细胞中 LTCC 膜密度的快速增加。FluoBar1 可能有助于解决以前关于细胞事件中 LTCC 动力学的棘手问题。