title: Inhibition of intracellular dipeptide hydrolysis uncovers large outward transport currents of the peptide transporter PEPT1 in Xenopus oocytes.
abstract: The "reversed transport mode" of electrogenic carriers is usually difficult to assess, as substrates are metabolized after reaching the cell, and the cytosolic surface is only accessible in special experimental settings such as giant-patch techniques. In the present experiments with the two-electrode voltage clamp, we demonstrate a unique feature of the peptide transporter PEPT1 that produces huge outward transport currents when oocytes are preloaded with hydrolysis-resistant dipeptides or when intracellular hydrolysis is prevented by aminopeptidase inhibition. A rapid intracellular degradation of dipeptides in oocytes and a parallel decline of outward currents were observed by analysis of amino acids in the cells. Dipeptide hydrolysis could almost completely be blocked by preincubation of oocytes with the aminopeptidase-inhibitor bestatin, itself a substrate of PEPT1. Dipeptide-driven outward currents of bestatin-treated oocytes remained stable over at least 10 min. Unexpectedly, the outward currents at a membrane potential of +60 mV were about five times higher than the corresponding inward currents measured before preloading at -60 mV under symmetrical conditions. The huge outward current was carried by PEPT1 and did not result from opening of potassium or chloride conductances in the membrane. Dipeptide-preloading of oocytes also increased inward currents evoked by substrates provided on the outside and equal substrate concentrations on both membrane surfaces in the absence of a pH gradient resulted in a linear current-voltage relation crossing the current axis at the origin. Our data and preliminary model calculations suggest a faster turnover rate of rPEPT1 in the presence of high substrate concentrations on the cytosolic surface.
authors.affiliation: Molecular Nutrition Unit, Technische Universität München, Freising, Germany. kottra@wzw.tum.de
authors.forename: Gabor
authors.initials: G
authors.lastname: Kottra
authors.forename: Isabelle
authors.initials: I
authors.lastname: Frey
authors.forename: Hannelore
authors.initials: H
authors.lastname: Daniel