STOICHIOMETRY AND PH-DEPENDENCE OF THE RABBIT PROTON-DEPENDENT OLIGOPEPTIDE TRANSPORTER PEPT1

1. The intestinal H+-coupled peptide transporter PepT1, displays a bro ad substrate specificity and accepts most charged and neutral di- and tripeptides. To study the proton-to-peptide stoichiometry and the depe ndence of the kinetic parameters on extracellular pH (pH(o)), rabbit P epT1 was expressed in Xenopus laevis oocytes and used for uptake studi es of radiolabelled neutral and charged dipeptides, voltage-clamp anal ysis and intracellular pH measurements. 2. PepT1 did not display the s ubstrate-gated anion conductances that have been found to be character istic of members of the Na+- and H+-coupled high-affinity glutamate tr ansporter family In conjunction with previous data on the ion dependen ce of PepT1, it can therefore be concluded that peptide-evoked charge fluxes of PepT1 are entirely due to H+ movement. 3. Neutral, acidic an d basic dipeptides induced intracellular acidification. The rate of ac idification, the initial rates of the uptake of radiolabelled peptides and the associated charge fluxes gave proton-substrate coupling ratio s of 1:1, 2:1 and 1:1 for neutral, acidic and basic dipeptides, respec tively. 4. Maximal transport of the neutral and charged dipeptides Gly -Leu, Gly-Glu, Gly-Lys and Ala-Lys occurred at pH(o) 5.5, 5.2, 6.2 and 5.8, respectively. The I-max values were relatively pH(o) independent but the apparent affinity (K-m(app)) values for these peptides were s hown to be highly pH(o) dependent. 5. Our data show that at physiologi cal pH (pH(o) 5.5-6.0) PepT1 prefers neutral and acidic peptides. The shift in transport maximum for the acidic peptide Gly-Glu to a lower p H value suggests that acidic dipeptides are transported in the protona ted form. The shift in the transport maxima of the basic dipeptides to higher pH values may involve titration of a sidechain on the transpor ter molecule (e.g. protonation of a histidine group). These considerat ions have led us to propose a model for coupled transport of neutral, acidic and basic dipeptides.