Stoichiometry and kinetics of the high-affinity H+-coupled peptide transporter PepT2

Proton-coupled peptide transporters mediate the absorption of a large varie ty of di- and tripeptides as well as peptide-like pharmacologically active compounds. We report a kinetic analysis of the rat kidney high-affinity pep tide transporter PepT2 expressed in Xenopus oocytes. By use of simultaneous radioactive uptake and current measurements under voltage-clamp condition, the charge to substrate uptake ratio was found to be close to 2 for both D -Phe-L-Ala and D-Phe-L-Glu, indicating that the H+:substrate stoichiometry is 2:1 and 3:1 for neutral and anionic dipeptides, respectively. The higher stoichiometry for anionic peptides suggests that they are transported in t he protonated form. For D-Phe-L-Lys, the charge:uptake ratio averaged 2.4 f rom pooled experiments, suggesting that Phe-Lys crosses the membrane via Pe pT2 either in its deprotonated (neutral) or its positively charged form, av eraging a H+:Phe-Lys stoichiometry of 1.4:1, These findings led to the over all con elusion that PepT2 couples transport of one peptide molecule to two H+; This is in contrast to the low-affinity transporter PepT1 that couples transport of one peptide to one H+. Quinapril inhibited PepT2-mediated cur rents in presence or in absence of external substrates, Oocytes expressing PepT2 exhibited quinapril-sensitive outward currents. In the absence of ext ernal substrate, a quinapril-sensitive proton inward current (proton leak) was also observed which, together with the observed pH-dependent PepT2-spec ific presteady-state currents (I-pss), indicates that at least one H+ binds to the transporter prior to substrate. PepT2 exhibited I-pss in response t o hyperpolarization at pH 6.5-8.0. However, contrary to previous observatio ns on various transporters, 1) no significant currents were observed corres ponding to voltage jumps returning from hyperpolarization, and 2) at reduce d extracellular pH, no significant I-pss were observed in either direction. Together with observed lower substrate affinities and decreased PepT2-medi ated currents at hyperpolarized V-m, our data are consistent with the conce pt that hyperpolarization exerts inactivation effects on the transporter wh ich are enhanced by low pH, Our studies revealed distinct properties of Pep T2, compared with PepT1 and other ion-coupled transporters.