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.

Automated summary

In this study, the human Peptide Transporter 1 (hPepT1) and its interactions with various substrates were comprehensively studied using solid supported membrane-based electrophysiology (SSME). The pH dependence of hPepT1 activity was observed, and the K-M and relative V-max values of different substrates were determined. Specific inhibition of hPepT1 by various inhibitors was also evaluated in a high throughput format.