Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors

Abstract

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 glutamate͞aspartate 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.aspartate ͉ hippocampal neuron ͉ neurotransmitter ͉ secretion ͉ transport I n addition to being an intermediate of primary metabolism in all biological cells, glutamate serves as the major excitatory amino acid neurotransmitter in the vertebrate central nervous system (1). As such, glutamate influences essentially all forms of behavior, including consciousness, sensory perception, motor control, and mood. Changes in the strength of connectivity at glutamatergic synapses in the form of long-term potentiation and long-term depression are considered to be the cellular mechanisms underlying learning and memory (2). In addition to its role in normal nervous system physiology, glutamate is also thought to be directly involved in neurologic damage occurring in stroke and neurodegenerative disorders, including AIDS-dementia complex, motor neuron disease, and Alzheimer's and Parkinson's diseases, through receptormediated toxicity (3).Despite its prominent role in normal and disease physiology, accurate and precise measurements of glutamate in living tissue are lacking. The concentration of glutamate in the cytoplasm and synaptic vesicles in neurons has been estimated by measurements of the amino acid in extracts (4). Extracellular glutamate concentrations have been measured by in vivo microdialysis techniques (5, 6). However, these techniques are limited in spatial and temporal resolution and are not suitable ...

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