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