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2018 - EAAT3 Investigated using SSM-based Electrophysiology

Icon 96SE   SURFE²R 96SE poster, Biophysics Annual Meeting 2018  logo pdf   (1.2 MB)

 Abstract:

The excitatory amino acid transporter 3 (EAAT3) is involved in the neuronal re-uptake of glutamate and plays a central role in the regulation of excitatory neurotransmission and
synaptic plasticity. EAAT3 also transports cysteine, necessary for the synthesis of glutathione and GABA. EAAT3 is expressed not only throughout the brain, but in many organs such as intestines, liver and heart. Here it seems to provide the main pathway of aspartate. Several connections of EAAT3 to severe neuronal disorders like epilepsy and schizophrenia have been described, as well as to metabolic disturbances concerning in the maintenance of aspartate and cysteine levels. This makes EAAT 3 not only an important target for functional research, but also a potential drug target.

Here we introduce a novel assay on EAAC1, a mouse homologue of EAAT3, using SSM (solid supported membrane)-based electrophysiology. SSM-based electrophysiology is a label-free electrical measuring method with very high sensitivity which enables the resolution of low turnover transport and even binding-events. Using the purified membrane of EAAC1 expressing CHO cells, we were able to determine substrate affinities and their interaction and to compare the effect of six known inhibitors directly with each other. We evaluated the assay stability and success rate. Furthermore, we were able to resolve substrate binding and to confirm the described anion conductance of the transporter.
Implementing SSM-based electrophysiology we were able to generate an efficient, robust and very flexible assay with a good throughout, which is an ideal tool for the biophysical and
pharmacological characterization of EAAC1 and even suitable for drug screening approaches.

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