title: Functional and structural determinants of reverse operation in the pH-dependent oligopeptide transporter PepT1.
abstract: The functional and structural basis of reverse operation of PepT1 has been studied in Xenopus oocytes expressing the wild-type and mutated forms of this protein. Using brief pulses from a negative holding potential, wild-type and Arg282 mutants exhibit outward currents in the presence of Gly-Gln. The reversal potential of these currents is affected by both pH and substrate concentration, confirming coupled transport in the wild type and in the mutants as well. Long-lasting voltage and current-clamp experiments show that the outward currents are only temporary, and reflect accumulation and/or depletion effects near the membrane. The ability to operate in reverse mode was confirmed in all isoforms by intracellular injection of substrate. The role of Arg282 and Asp341 in the reverse transport was also investigated using charged substrates. Positive Lys-Gly (but not Gly-Lys) showed enhanced transport currents in the Arg282 mutants. In contrast, negative Gly-Asp and Asp-Gly elicited modest currents in all isoforms.
authors.affiliation: Laboratory of Cellular and Molecular Physiology, Dept. of Biotechnology and Molecular Sciences, University of Insubria, Via Dunant 3, 21100, Varese, Italy.
authors.forename: Maria Daniela
authors.initials: MD
authors.lastname: Renna
authors.forename: Ayodele Stephen
authors.initials: AS
authors.lastname: Oyadeyi
authors.forename: Elena
authors.initials: E
authors.lastname: Bossi
authors.forename: Gabor
authors.initials: G
authors.lastname: Kottra
authors.forename: Antonio
authors.initials: A
authors.lastname: Peres
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D004151:Dipeptides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D000074011:Peptide Transporter 1; D011817:Rabbits; D013379:Substrate Specificity; D027981:Symporters / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014982:Xenopus laevis / Q000196:embryology
chemical_list: D004151:Dipeptides; D000074011:Peptide Transporter 1; D027981:Symporters; C437951:lysylglycine; C039788:glycylglutamine; C017033:aspartylglycine