Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors.
Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery.
Okumoto
Sakiko
S
Department of Plant Biology, Carnegie Institution of Washington, 260 Panama Street, Stanford, CA 94305, USA.
Looger
Loren L
LL
Micheva
Kristina D
KD
Reimer
Richard J
RJ
Smith
Stephen J
SJ
Frommer
Wolf B
WB
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D016296:Mutagenesis; D049329:Nanostructures; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D016716:PC12 Cells; D010957:Plasmids / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid