1. High-throughput development of a hybrid-type fluorescent glutamate sensor for analysis of synaptic transmission.
- Author
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Takikawa K, Asanuma D, Namiki S, Sakamoto H, Ariyoshi T, Kimpara N, and Hirose K
- Subjects
- Animals, Fluorescent Dyes analysis, Glutamic Acid analysis, High-Throughput Screening Assays, Humans, Models, Molecular, Optical Imaging, Synapses metabolism, Fluorescent Dyes metabolism, Glutamic Acid metabolism, Receptors, AMPA metabolism, Synaptic Transmission
- Abstract
Fluorescent sensors are powerful tools for visualizing cellular molecular dynamics. We present a high-throughput screening system, designated hybrid-type fluorescence indicator development (HyFInD), to identify optimal position-specific fluorophore labeling in hybrid-type sensors consisting of combinations of ligand-binding protein mutants with small molecular fluorophores. We screened sensors for glutamate among hybrid molecules obtained by the reaction of four cysteine-reactive fluorescence probes with a set of cysteine-scanning mutants of the 274 amino acid S1S2 domain of AMPA-type glutamate receptor GluA2 subunit. HyFInD identified a glutamate-responsive probe (enhanced glutamate optical sensor: eEOS) with a dynamic range >2400โ%, good photostability, and high selectivity. When eEOS was specifically tethered to neuronal surfaces, it reliably visualized the spatiotemporal dynamics of glutamate release at single synapses, revealing synapse-to-synapse heterogeneity of short-term plasticity., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
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