Transport of charged substrates by the intestinal peptide carrier pept1 expressed in xenoupus laevis ooctyes

The H⁺/peptide cotransporter in the apical membrane of intestinal epithelial cells mediates uptake of di- and tripeptides and selected peptidomimetics Based on the 20 amino acids there are 400 possible dipepti-des and 8000 different tripeptides which could serve as substrates for the transport system including a large variety of substrates carrying net negative or positive charge. A fundamental question that has not been studied yet is whether and how the transporter translocates differently charged substrates. We have employed flux studies with selected radiolabeled dipeptides and the two electrode voltage clamp technique in ooytes expressing the rabbit intestinal peptide transporter PepT1 to gain informations on substrate dependent changes in membrane potential and the substrate flux coupling to proton translocation. In addition we have assessed substrate transport-coupled proton influx into oocytes by measuring pHjn changes with the dual emission pH sensitive indicator dextran-SNARF!. With a series of neutral and charged glycyl-dipeptides we demonstrate that the transporter is capable of translocating all substrates by an electrogenic process regardless of the substrates net charge. Whereas the Km values for substrate interaction with PepT1 are not affected by pH in case of zwirterionic substrates, affinity changes pH dependent for charged dipeptides. The driving force is provided by the inside negative transmembrane electrical potential and in case of neutral and anionic substrates additionally by the transmembrane H+ gradient. Electrogenicity of transport of neutral and charged substrates is achieved by different flux coupling ratios (0, 1 or 2) for H⁺ or H₃O⁺ cotransport resulting in all cases in net transfer of one positive charge during a cycle of substrate translocation.