The oppositely charged residues Arg282 and Asp341 in the transmembrane domains 7 and 8 of the intestinal peptide transporter PEPT1 have been found to form an electrostatic barrier in the absorption pathway. We measured presteady-state and steady-state currents in Xenopus oocytes expressing wildtype (WT) and mutated PEPT1 and show here that mutations of these residues affect the passage of the cotransported protons. Substitution of Arg282 with neutral or negative residues produced a shift of presteady-state charge movement towards less negative potentials with respect to the WT form. In addition, the inward rectification observed in WT disappeared and large steady outward currents were seen at small negative and positive membrane potentials. Conversely, replacement of Asp341 with Arg reduced both presteady-state and transport currents without affecting the surface expression and produced a negative shift of the charge movement properties. The double mutant Arg282Asp+Asp341Arg was only weakly expressed in the cell membrane and thus the properties of its presteady state currents could not reliably be investigated. In summary, removal or reversal of the positive field generated by Arg282 caused a negative distortion to the membrane potential profile, producing an increase in the field strenght effectively acting on the inward charge movement. To reach the same level of electrical field acting on the charge movement the intracellular voltage must compensate for this alteration and be set to less negative values. In contrast, the mutation Asp341Arg adds a positive component to the membrane potential profile and must be compensated by an increased membrane negativity.

Figure 1