Electrophysiological analysis of the function of the mammalian renal peptide transporter expressed in Xenopus laevis oocytes.
1. To gain information on the mode of operation of the renal proton-coupled peptide transporter PepT2, voltage clamp studies were performed in Xenopus laevis oocytes expressing the rabbit renal PepT2. 2. Using differently charged glycyl-dipeptides we show that PepT2 translocates these dipeptides by an electrogenic pH-dependent process that is essentially independent of the substrate net charge. The apparent substrate affinities are in the micromolar range (2-50 microM) between pH 5.5 and 7.4 and membrane potentials of +/- 0 to -50 mV. 3. Maximal substrate-evoked inward currents (Imax) are affected by membrane voltage (Vm) and extracellular pH (pHo). Potential-dependent interactions of H+/H3O+ with PepT2 seem to be mediated by a single low affinity binding site and PepT2 remains pH dependent at all voltages. 4. The effects of voltage on apparent Imax and substrate affinity display an inverse relationship. As Vm is altered from -50 to -150 mV substrate affinities decrease 10- to 50-fold whereas apparent Imax increases almost 10-fold. 5. Even at saturating H+/H3O+ and dipeptide concentrations the I-V curves did not show saturation at negative membrane potentials, suggesting that other steps in the reaction cycle and not the ligand affinity changes are rate limiting. These are possibly the conformational changes of the empty and/or loaded transporters. 6. These findings demonstrate that not only substrate affinities but also other kinetic characteristics of PepT2 differ markedly from those of the intestinal peptide transporter isoform PepT1.
Amasheh
S
S
Institute of Animal Physiology, University of Giessen, Germany.
Wenzel
U
U
Weber
W M
WM
Clauss
W
W
Daniel
H
H
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000502:physiology*; D004558:Electric Stimulation; D004594:Electrophysiology; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D007473:Ion Channels; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2