Genetically Encoded Glutamate Indicators with Altered Color and Topology,ACS Chemical Biology

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Genetically Encoded Glutamate Indicators with Altered Color and Topology
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2018-01-08 00:00:00 , DOI: 10.1021/acschembio.7b01085
Jiahui Wu ₁ , Ahmed S. Abdelfattah ₁ , Hang Zhou ₁ , Araya Ruangkittisakul ₂ , Yong Qian ₁ , Klaus Ballanyi ₂ , Robert E. Campbell ₁

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Glutamate is one of the 20 common amino acids and of utmost importance for chemically mediated synaptic transmission in nervous systems. To expand the color palette of genetically encoded indicators for glutamate, we used protein engineering to develop a red intensity-based glutamate-sensing fluorescent reporter (R-iGluSnFR1). Manipulating the topology of R-iGluSnFR1, and a previously reported green fluorescent indicator, led to the development of noncircularly permutated (ncp) variants. R- and R^ncp-iGluSnFR1 display glutamate affinities of 11 μM and 0.9 μM, respectively. We demonstrate that these glutamate indicators are functional when targeted to the surface of HEK-293 cells. Furthermore, we show that G^ncp-iGluSnFR enabled reliable visualization of extrasynaptic glutamate in organotypic hippocampal slice cultures, while R-iGluSnFR can reliably resolve action potential-evoked glutamate transients by electrical field stimuli in cultures of dissociated hippocampal neurons.

中文翻译：

基因编码的谷氨酸盐指示剂，具有改变的颜色和拓扑

谷氨酸是20种常见氨基酸之一，对于神经系统中化学介导的突触传递极为重要。为了扩展谷氨酸的遗传编码指示剂的调色板，我们使用了蛋白质工程技术来开发基于红色强度的谷氨酸感应荧光报告基因（R-iGluSnFR1）。操纵R-iGluSnFR1的拓扑结构和先前报道的绿色荧光指示剂，导致了非圆形排列（ncp）变体的发展。R-和R ^ncp -iGluSnFR1显示的谷氨酸亲和力分别为11μM和^0.9μM。我们证明这些谷氨酸指示剂针对HEK-293细胞的表面时功能。此外，我们证明了G ^ncp-iGluSnFR能够可靠地观察器官型海马切片培养物中突触外谷氨酸，而R-iGluSnFR可以通过电场刺激在离解的海马神经元培养物中可靠地分辨动作电位诱发的谷氨酸瞬变。

更新日期：2018-01-08

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