Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology

Alexander Körner; Andre Bazzone; Maximilian Wichert; Maria Barthmes; Srujan Kumar Dondapati; Niels Fertig; Stefan Kubick

doi:10.1016/j.bioelechem.2023.108573

Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology

Bioelectrochemistry. 2024 Feb:155:108573. doi: 10.1016/j.bioelechem.2023.108573. Epub 2023 Sep 16.

Authors

Alexander Körner¹, Andre Bazzone², Maximilian Wichert³, Maria Barthmes², Srujan Kumar Dondapati⁴, Niels Fertig², Stefan Kubick⁵

Affiliations

¹ Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 14476 Potsdam, Germany; Institute of Biotechnology, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany.
² Nanion Technologies GmbH, Ganghoferstr. 70a, 80339 Munich, Germany.
³ Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 14476 Potsdam, Germany; Freie Universität Berlin, Institute of Chemistry and Biochemistry - Biochemistry, 14195 Berlin, Germany.
⁴ Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 14476 Potsdam, Germany. Electronic address: Srujan.Dondapati@izi-bb.fraunhofer.de.
⁵ Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 14476 Potsdam, Germany; Freie Universität Berlin, Institute of Chemistry and Biochemistry - Biochemistry, 14195 Berlin, Germany; Faculty of Health Sciences, Joint Faculty of the Brandenburg University of Technology Cottbus - Senftenberg, The Brandenburg Medical School Theodor Fontane and the University of Potsdam, Germany.

PMID: 37748262
DOI: 10.1016/j.bioelechem.2023.108573

Abstract

The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported membrane-based electrophysiology (SSME). Using hPepT1-containing vesicles, we could not identify any peptide induced pre-steady-state currents, indicating that the recorded peak currents reflect steady-state transport. Electrogenic co-transport of H⁺/glycylglycine (GlyGly) was observed across a pH range of 5.0 to 9.0. The pH dependence is described by a bell-shaped activity curve and two pK values. K_M and relative V_max values of various canonical and non-canonical peptide substrates were contextualized with current mechanistic understandings of hPepT1. Finally, specific inhibition was observed for various inhibitors in a high throughput format, and IC50 values are reported. Taken together, these findings contribute to promoting the design and analysis of pharmacologically relevant substances.

Keywords: Biosensor; High‐throughput screening (HTS); Membrane transport; Peptide Transporter 1 (PepT1); Pharmacology; Solid Supported Membrane-based Electrophysiology (SSME).

MeSH terms

Electrophysiology
Humans
Kinetics
Peptide Transporter 1
Peptides
Symporters* / metabolism

Substances

Symporters
Peptide Transporter 1
Peptides