Protonation State of a Histidine Residue in Human Oligopeptide Transporter 1 (hPEPT1) Regulates hPEPT1-Mediated Efflux Activity

To clarify the role of an amino acid residue in the pH-dependent efflux process in Chinese hamster ovary (CHO) cells expressing the human oligopeptide transporter hPEPT1 (CHO/hPEPT1), we determined the effect of extracellular pH on the hPEPT1-mediated efflux process. The efflux of glycylsarcosine (Gly-Sar), a typical substrate for hPEPT1, was determined using an infinite dilution method after cells were preloaded with [H-3]-Gly-Sar. The efflux of [H-3]-Gly-Sar was stimulated by 5 mM unlabeled hPEPT1 substrates in the medium. This trans-stimulation phenomenon showed that hPEPT1 mediated the efflux of [H-3]-Gly-Sar from CHO/hPEPT1 and that hPEPT1 is a bi-directional transporter. We then determined the effect of extracellular pH (varying from 8.0 to 3.5) on the efflux activity. The efflux activity by hPEPT1 decreased with the decrease in extracellular pH. The Henderson-Hasselbalch-type equation, which fitted well to the pH-profile of efflux activity, indicated that a single amino acid residue with a pK(a) value of approximately 5.7 regulates the efflux activity. The pH-profile of the efflux activity remained almost unchanged irrespective of the proton gradient across the plasma membrane. In addition, the chemical modification of the histidine residue with diethylpyrocarbonate completely abolished the efflux activity from cells, which could be prevented by the presence of 10 mM Gly-Sar. These data indicate that the efflux process of hPEPT1 is also regulated in a pH-dependent manner by the protonation state of a histidine residue located at or near the substrate recognition site facing the extracellular space.

Automated summary

The study revealed that the pH-dependent efflux process mediated by hPEPT1 in CHO/hPEPT1 cells is regulated by a single amino acid residue with a pKa value around 5.7. The efflux activity by hPEPT1 decreases as extracellular pH decreases, showing that the protonation state of a histidine residue near the substrate recognition site plays a crucial role in regulating the efflux process. These findings demonstrate that hPEPT1 is a bi-directional transporter and that its efflux activity is modulated in a pH-dependent manner.