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

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.
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Authors AB, MB and NF are employed by Nanion Technologies GmbH, the developer of the SURFE(2)R instruments.
(Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)