The extracellular pH dependency of transport activity by human oligopeptide transporter 1 (hPEPT1) expressed stably in Chinese hamster ovary (CHO) cells: a reason for the bell-shaped activity versus pH.
Human oligopeptide transporter (hPEPT1) translocates di/tri-peptide by coupling to movement of proton down the electrochemical gradient. This transporter has the characteristics that the pH-profile of neutral dipeptide transport shows a bell-shaped curve with an optimal pH of 5.5. In the present study, we examined the reason for the decrease in the acidic region with hPEPT1-transfected CHO cells stably oeverexpressing hPEPT1 (CHO/hPEPT1). The pH profile of the transport activity vs. pH was measured in the presence of nigericin/monensin. Under this condition, the inwardly directed proton concentration gradient was dissipated while the membrane potential remained. As pH increased the activity increased, and the Henderson-Hasselbalch equation with a single pKa was fitted well to the activity curve. The pKa value was estimated to be 6.7+/-0.2. This value strongly suggests that there is a key amino acid residue, which is involved in pH regulation of transport activity. To identify the key amino acid residue, we examined the effects of various chemical modifications on pH-profile of the transport activity. Modification of carboxyl groups or hydroxyl groups had no significant influence on the pH-profile, whereas a chemical modification of histidine residue with diethylpyrocarbonate (DEPC) completely abolished the transport activity in CHO/hPEPT1 cells. On the other hand, this abolishment was almost prevented by the presence of 10 mM Gly-Sar. This protection was observed only in the presence of the substrate of hPEPT1, indicating that the histidine residue is located at the substrate recognition site. The pH-profile of the transport activity in CHO/hPEPT1 cells treated with DEPC in the presence of 10 mM Gly-Sar also showed a bell-shape similar to that in non-treated CHO/hPEPT1 cells. These data stressed that the histidine residue located at or near the substrate binding site is involved in the pH regulation of transport activity.
Fujisawa
Yuki
Y
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Tateoka
Ryoko
R
Nara
Toshifumi
T
Kamo
Naoki
N
Taira
Takahiro
T
Miyauchi
Seiji
S
D000465:Algorithms; D000818:Animals; D016466:CHO Cells; D018938:Caco-2 Cells; D006224:Cricetinae; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D005110:Extracellular Space / Q000737:chemistry / Q000378:metabolism*; D005822:Genetic Vectors; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D000074011:Peptide Transporter 1; D013379:Substrate Specificity; D027981:Symporters / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*
D004151:Dipeptides; D007202:Indicators and Reagents; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; D006639:Histidine; D004047:Diethyl Pyrocarbonate