Genetically Encoded Halide Sensor-Based Fluorescent Assay for Rapid Screening of Glutamate Transport and Inhibition

ACS Sensors
Laura Zielewicz, Christof Grewer

Abstract

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system. Excitatory amino acid transporters (EAATs) are a family of transmembrane transporters responsible for glutamate uptake into cells, and their malfunction is related to a variety of diseases, including neurodegenerative diseases and stroke. Screening for and developing inhibitors of EAATs as well as related transporters is a significant field of study for biomedical and pharmaceutical applications. Rapid, high-throughput methods are critical for the study of glutamate transporters, and fluorescent methods are appealing for this purpose as compared to more traditional electrophysiological methods. In this study, we present a method for studying glutamate transporters and inhibitors by utilizing a mutated version of a yellow fluorescent protein (YFP) highly sensitive to quenching by anions (mClY). We applied this YFP variant to fluorescent imaging of anion flux in HEK293 cells caused by transiently expressed excitatory amino acid carrier 1 (EAAC1) and excitatory amino acid transporter 2 (EAAT2) and its inhibition by competitive blockers. This method enables rapid identification of inhibitors and, potentially, activators of EAAT function, whic...Continue Reading

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