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2021 - Functional Characterization of SLC Transporters Using Solid Supported Membranes

Icon N1 SURFE2R N1 Chapter in Biophysics of Membrane Proteins (2021)

Authors:
Bazzone A., Barthmes M.

Book:
Part of the Methods in Molecular Biology book series in Biophysics of Membrane Proteins. doi: 10.1007/978-1-0716-0724-4_4


Abstract:

Here, we present a protocol for the functional characterization of the H+-coupled human peptide transporter PepT1 and sufficient notes to transfer the protocol to the Na+-coupled sugar transporter SGLT1, the organic cation transporter OCT2, the Na+/Ca2+ exchanger NCX, and the neuronal glutamate transporter EAAT3.

The assay was developed for the commercially available SURFE2R N1 instrument (Nanion Technologies GmbH) which applies solid supported membrane (SSM)-based electrophysiology. This technique is widely used for the functional characterization of membrane transporters with more than 100 different transporters characterized so far. The technique is cost-effective, easy to use, and capable of high-throughput measurements.

SSM-based electrophysiology utilizes SSM-coated gold sensors to physically adsorb membrane vesicles containing the protein of interest. A fast solution exchange provides the substrate and activates transport. For the measurement of PepT1 activity, we applied a peptide concentration jump to activate H+/peptide symport. Proton influx charges the sensor. A capacitive current is measured reflecting the transport activity of PepT1. Multiple measurements on the same sensor allow for comparison of transport activity under different conditions. Here, we determine EC50 for PepT1-mediated glycylglycine transport and perform an inhibition experiment using the specific peptide inhibitor Lys[Z(NO2)]-Val.


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