TRANSPORT OF CHARGED DIPEPTIDES BY THE INTESTINAL H+ PEPTIDE SYMPORTER PEPT1 EXPRESSED IN XENOPUS-LAEVIS OOCYTES/

The cloned intestinal peptide transporter is capable of electrogenic H +-coupled cotransport of neutral di- and tripeptides and selected pept ide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the char acteristics of transport of differently charged glycylpeptides. Transp ort function of PepT1 was assessed by flux studies employing a radiola beled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all sub strates by an electrogenic process that follows Michaelis Menten kinet ics. Whereas the apparent K-0.5 value of a zwitterionic substrate is o nly moderately affected by alterations in pH or membrane potential, K- 0.5 values of charged substrates are strongly dependent on both, pH an d membrane potential. Whereas the affinity of the anionic dipeptide in creased dramatically by lowering the pH, a cationic substrate shows on ly a weak affinity for PepT1 at all pH values (5.5-8.0). The driving f orce for uptake is provided mainly by the inside negative transmembran e electrical potential. In addition, affinity for proton interaction w ith PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest that uptake of the zwitterionic form of a charged substrate c ontributes to overall transport and that consequently the stoichiometr y of the flux-coupling ratios for peptide: H+/H2O+ cotransport may var y depending on pH.