Transport function of the renal peptide carrier rPEPT2 expressed in xenopus laevis oocytes

The H⁺-coupled electrogenic high affinity peptide transporter (rPepT2) in the apical membrane of tubular cells mediates uptake of filtered short chain peptides and xenobiotics with a peptide backbone. The transporter has recently been cloned from a rabbit kidney cortex cDNA library but has not been characterized with respect to peptide transport function. We therefore studied rPepT.2 carrier function in flux studies using selected radiolabeled dipeptides and by the two electrode voltage clamp technique in oocytes injected with 5ng of the transporters cRNA Steady state current-voltage (I-V) relationships were measured in the absence and the presence of different peptides with the membrane potential in oocytes clamped to -60 mV. Results: Uptake of -³H-glycytL-glutamine (Gly-GIn) and ³H-D-phenyl-L-alanine (D-Phe-Ala) into oocytes increased 6-fold by reducing pH from 7.4 to 5.5 Uptake as a function of dipeptide concentration revealed saturation kinetics with app K_m values of 52 ± 5 nM (Gly-GIn) and 83 ±9 nM (D-Phe-Ala) and similar Vmax values. Competition experiments established that di- and tripeptides interact m a stereospecific manner with the substrate binding site of rPepT2. At pH 6.0, neutral, anionic and cationic dipeptides showed similar affinity constants between 45 and 80 μM When current was measured in voltage clamped oocytes, all substrates tested (including charged dipeptides) caused inward currents of similar magnitudes I-V relationships displayed characteristic pH dependencies for all substrates. Uptake of Gly-GIn assessed by currents generated and by influx of the radiolabeled substrate correlates significantly and predicts a flux coupling ratio of 11 (peptidei:H⁺) at negative membrane potentials.