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Free keywords:
Membrane protein; Solid supported membrane; CHO cells; Glutamate transport; Anion conductance; Drug screening
Abstract:
A rapid and robust electrophysiological assay based on solid supported membranes (SSM) for the murine neuronal glutamate transporter mEAAC1 is presented. Measurements at different concentrations revealed the EAAC1 specific affinities for l-glutamate (Km = 24 μM), l-aspartate (Km = 5 μM) and Na+ (Km = 33 mM) and an inhibition constant Ki for dl-threo-β-benzyloxyaspartic acid (TBOA) of 1 μM. Inhibition by 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) was not purely competitive with an IC50 of 13 μM. Experiments using SCN− concentration jumps yielded large transient currents in the presence of l-glutamate showing the characteristics of the glutamate-gated anion conductance of EAAC1. Thus, SSM-based electrophysiology allows the analysis of all relevant transport modes of the glutamate transporter on the same sample.
K+ and Na+ gradients could be applied to the transporter. Experiments in the presence and absence of Na+ and K+ gradients demonstrated that the protein is still able to produce a charge translocation when no internal K+ is present. In this case, the signal amplitude is smaller and a lower apparent affinity for l-glutamate of 144 μM is found.
Finally the assay was adapted to a commercial fully automatic system for SSM-based electrophysiology and was validated by determining the substrate affinities and inhibition constants as for the laboratory setup. The combination of automatic function and its ability to monitor all transport modes of EAAC1 make this system an universal tool for industrial drug discovery.
