Genetically Encoded Glutamate Indicators with Altered Color and Topology
| Autor: | Yong Qian, Hang Zhou, Ahmed S. Abdelfattah, Robert E. Campbell, Jiahui Wu, Klaus Ballanyi, Araya Ruangkittisakul |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Protein Conformation Hippocampal slice Color Glutamic Acid Neurotransmission Hippocampal formation Protein Engineering Topology Hippocampus Biochemistry Rats Sprague-Dawley 03 medical and health sciences 0302 clinical medicine Animals Humans Fluorescent Dyes chemistry.chemical_classification Fluorescent reporter Glutamate receptor General Medicine Protein engineering Fluorescence Amino acid Luminescent Proteins HEK293 Cells 030104 developmental biology chemistry Mutation Molecular Medicine 030217 neurology & neurosurgery Protein Binding |
| Zdroj: | ACS Chemical Biology. 13:1832-1837 |
| ISSN: | 1554-8937 1554-8929 |
| Popis: | 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 Rncp-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 Gncp-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. |
| Databáze: | OpenAIRE |
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