title: High-throughput development of a hybrid-type fluorescent glutamate sensor for analysis of 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 >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.
authors.affiliation: Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo (Japan).
authors.forename: Kenji
authors.initials: K
authors.lastname: Takikawa
authors.forename: Daisuke
authors.initials: D
authors.lastname: Asanuma
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Namiki
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Sakamoto
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Ariyoshi
authors.forename: Naoya
authors.initials: N
authors.lastname: Kimpara
authors.forename: Kenzo
authors.initials: K
authors.lastname: Hirose
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D018698:Glutamic Acid / Q000032:analysis / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D008958:Models, Molecular; D061848:Optical Imaging; D018091:Receptors, AMPA / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism; D009435:Synaptic Transmission
chemical_list: D005456:Fluorescent Dyes; D018091:Receptors, AMPA; D018698:Glutamic Acid
keywords: fluorescent probes; high-throughput screening; neurotransmitters