ELECTROPHYSIOLOGICAL ANALYSIS OF THE FUNCTION OF THE MAMMALIAN RENAL PEPTIDE TRANSPORTER EXPRESSED IN XENOPUS-LAEVIS OOCYTES

1. To gain information on the mode of operation of the renal proton-co upled peptide transporter PepT2, voltage clamp studies were performed in Xenopus laevis oocytes expressing the rabbit renal PepT2. 2. Using differently charged glycyl-dipeptides we show that PepT2 translocates these dipeptides by an electrogenic pH-dependent process that is essen tially independent of the substrate net charge. The apparent substrate affinities are in the micromolar range (2-50 mu M) between pH 5.5 and 7.4 and membrane potentials of +/-0 to -50 mV. 3. Maximal substrate-e voked inward currents (I-max) are affected by membrane voltage (V-m) a nd extracellular pH (pH(o)). Potential-dependent interactions of H+/H3 O+ with PepT2 seem to be mediated by a single low affinity binding sit e and PepT2 remains pH dependent at all voltages. 4. The effects of vo ltage on apparent I-max and substrate affinity display an inverse rela tionship. As V-m is altered. from -50 to -150 mV substrate affinities decrease 10- to 50-fold whereas apparent I-max increases almost 10-fol d. 5. Even at saturating H+/H3O+ and dipeptide concentrations the I-V curves did not show saturation at negative membrane potentials, sugges ting that other steps in the reaction cycle and not the ligand affinit y changes are rate limiting. These are possibly the conformational cha nges of the empty and/or loaded transporters. 6. These findings demons trate that not only substrate affinities but also other kinetic charac teristics of PepT2 differ markedly from those of the intestinal peptid e transporter isoform PepT1.