Transport of compounds by the proton-coupled epithelial peptide transporter PepT1 is known to be dependent on the transmembrane pH gradient and the membrane potential.

In this study, we have investigated the effects of changing the intracellular pH on the basal and trans-stimulated efflux of a known PepT1 substrate, tritiated D-Phe-L-Gln. Flux experiments were performed at an acidic (5.5) and neutral (7.4) pH and the intracellular pH of the oocytes was acidified (Quick et al. 2001) in the presence and absence of a membrane potential using increasing concentrations of extracellular sodium and potassium acetate, respectively.

For each experimental condition, five PepT1-expressing oocytes were injected with [³H] D-Phe-L-Gln (9.2 nl; 30 µM) and incubated in 100 µl uptake medium (pH 5.5 or pH 7.4) at 20 °C for 60 min. Aliquots (70 µl) of the bathing medium were collected and the radioactive content quantified. Addition of increasing concentrations of unlabelled D-Phe-L-Gln (0-20 mM) allowed affinity estimates for the efflux of D-Phe-L-Gln to be obtained. Composition of uptake media was (mM): NaCl (95), KCl (2), CaCl₂ (1), MgCl₂ (0.1), Hepes (20) or MES (20), pH adjusted with Tris. The PepT1-expressing oocytes used were all 4-5 days post-cRNA injection. Data from example experiments are shown in Fig. 1A and B.

Upon extracellular acidification, the extent of maximal efflux by external D-Phe-L-Gln decreases from 118.64 ± 5.11 fmol oocyte^-1 h^-1 at pH 7.4 to 97.88 ± 7.74 fmol oocyte^-1 h^-1 at pH 5.5 and the affinity decreases from 0.10 ± 0.02 mM at pH 7.4 to 0.36 ± 0.07 mM at pH 5.5 (n = 3/4, ± S.E.M., P < 0.05, Student’s t test). In addition, intracellular acidification with 25 mM acetate results in a dramatic increase in affinity when pH_o = 5.5 (0.36 ± 0.07 mM in comparison with 0.09 ± 0.04 mM).The data from these experiments and others where the cells have been depolarised at each pH (data not shown) are consistent with most but not all aspects of a previously proposed model (Temple et al. 1996) for wild-type PepT1.We thank M.A. Hediger for the PepT1 clone and the MRC and Wellcome Trust for financial support.

Figure 1. The effects of intracellular acidification (using increasing concentrations of acetate) on trans-stimulation of D-Phe-L-Gln efflux.

Quick, M., Loo, D.D.F. & Wright, E.M. (2001). J. Biol. Chem. 276, 1728-1734.

Temple, C.S., Bailey, P.D., Bronk, J.R. & Boyd, C.A.R. (1996). J. Physiol. 494, 795-808. abstract