Search form

TitleGenetically Encoded Glutamate Indicators with Altered Color and Topology.
Publication TypeJournal Article
Year of Publication2018
AuthorsWu, Jiahui, Ahmed S. Abdelfattah, Hang Zhou, Araya Ruangkittisakul, Yong Qian, Klaus Ballanyi, and Robert E. Campbell
JournalACS Chem Biol
Date Published2018 Jan 22
ISSN1554-8937
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 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.

DOI10.1021/acschembio.7b01085
Alternate JournalACS Chem. Biol.
PubMed ID29308878