Genetically Encoded Glutamate Indicators with Altered Color and Topology
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 fluores...
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Published in | ACS chemical biology Vol. 13; no. 7; pp. 1832 - 1837 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
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American Chemical Society
20.07.2018
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Abstract | 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. |
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AbstractList | 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. 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 -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 -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. |
Author | Ballanyi, Klaus Campbell, Robert E Qian, Yong Abdelfattah, Ahmed S Ruangkittisakul, Araya Zhou, Hang Wu, Jiahui |
AuthorAffiliation | Department of Chemistry Department of Physiology |
AuthorAffiliation_xml | – name: Department of Physiology – name: Department of Chemistry |
Author_xml | – sequence: 1 givenname: Jiahui surname: Wu fullname: Wu, Jiahui organization: Department of Chemistry – sequence: 2 givenname: Ahmed S surname: Abdelfattah fullname: Abdelfattah, Ahmed S organization: Department of Chemistry – sequence: 3 givenname: Hang surname: Zhou fullname: Zhou, Hang organization: Department of Chemistry – sequence: 4 givenname: Araya surname: Ruangkittisakul fullname: Ruangkittisakul, Araya organization: Department of Physiology – sequence: 5 givenname: Yong surname: Qian fullname: Qian, Yong organization: Department of Chemistry – sequence: 6 givenname: Klaus surname: Ballanyi fullname: Ballanyi, Klaus organization: Department of Physiology – sequence: 7 givenname: Robert E orcidid: 0000-0003-0604-092X surname: Campbell fullname: Campbell, Robert E email: robert.e.campbell@ualberta.ca organization: Department of Chemistry |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/29308878$$D View this record in MEDLINE/PubMed |
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Title | Genetically Encoded Glutamate Indicators with Altered Color and Topology |
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