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Functional Characterization of SLC Transporters Using Solid Supported Membranes

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Biophysics of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2168))

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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Authors and Affiliations

  1. Nanion Technologies GmbH, Munich, Germany

    Andre Bazzone & Maria Barthmes

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  1. Andre Bazzone
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  2. Maria Barthmes
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Correspondence to Andre Bazzone .

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Editors and Affiliations

  1. School of Clinical and Applied Sciences, Leeds Beckett University, Leeds, UK

    Vincent L. G. Postis

  2. Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences; Molecular and Integrative Biosciences, Faculty of Biological and Environmental Sciences, University of Leeds, Leeds, UK; University of Helsinki, Helsinki, Finland

    Adrian Goldman

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Bazzone, A., Barthmes, M. (2020). Functional Characterization of SLC Transporters Using Solid Supported Membranes. In: Postis, V.L.G., Goldman, A. (eds) Biophysics of Membrane Proteins. Methods in Molecular Biology, vol 2168. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0724-4_4

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  • DOI: https://doi.org/10.1007/978-1-0716-0724-4_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0723-7

  • Online ISBN: 978-1-0716-0724-4

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