The epithelial di- and tri-peptide transporter PepT1 couples substrate and proton movement in such a way that transport is powered both by the physiological proton gradient across the apical membrane and by the membrane potential (Temple et al. 1995). Previously, uptake of the neutral dipeptide D-Phe-L-Gln into R282E-PepT1-expressing oocytes at pH_out 5.5 had revealed a functional transporter (Meredith, 2003), which has now been further characterised. Here we report that the site-directed mutagenesis of this arginine residue abolishes the proton coupling of peptide transport.

Arginine₂₈₂ in rabbit PepT1 was mutated to a glutamate using a PCR-based protocol (Quikchange, Stratagene) and confirmed by DNA sequencing to produce R282E-PepT1. Uptake experiments into PepT1-expressing Xenopus laevis oocytes were performed as previously described (Meredith et al. 2000). Data are means ± S.E.M.

Uptake of D-Phe-L-Gln into oocytes expressing R282E-PepT1 was found to be unaffected by extracellular acidification (1.1 ± 0.4-fold increase, n = 2), unlike for the wild-type PepT1, which was stimulated by reducing pH_out from 7.4 to 5.5 by 2.4 ± 0.3-fold (P < 0.05, Student’s paired t test, n = 4). To investigate whether this was due to peptide uptake no longer being coupled to the proton electrochemical gradient, the ability of the oocytes to perform concentrative uptake at pH_out 5.5 was measured, as shown in Fig. 1. As can be seen, R282E-PepT1-expressing oocytes were not able to concentrate D-Phe-L-Gln, unlike those expressing WT-PepT1 which achieved approximately 4-fold concentration above the extracellular level after 4 h.

Taken together, the data that D-Phe-L-Gln uptake by R282E-PepT1 is not stimulated by external acidification nor can R282E-PepT1-expressing oocytes concentrate dipeptide above the external concentration strongly suggest that the proton and dipeptide translocation have been uncoupled, and that R282E-PepT1 behaves as a facilitated diffusion system.

This work is generously funded by the Wellcome Trust.