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High‐Throughput Development of a Hybrid‐Type Fluorescent Glutamate Sensor for Analysis of Synaptic Transmission
Author(s) -
Takikawa Kenji,
Asanuma Daisuke,
Namiki Shigeyuki,
Sakamoto Hirokazu,
Ariyoshi Tetsuro,
Kimpara Naoya,
Hirose Kenzo
Publication year - 2014
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201407181
Subject(s) - glutamate receptor , fluorophore , fluorescence , ampa receptor , biophysics , synapse , chemistry , cysteine , biochemistry , biology , neuroscience , receptor , quantum mechanics , enzyme , physics
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 >2400 %, 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.