lysylglycine and glycylglutamine

The oligopeptide transporter PepT1 is a protein found in the membrane of the cells of the intestinal walls, and represents the main route through which proteic nutrients are absorbed by the organism. Along the polypeptidic chain of this protein, two oppositely charged amino acids, an arginine in position 282 and an aspartate in position 341 of the sequence, have been hypothesised to form a barrier in the absorption pathway. In this paper we show that appropriate mutations of these amino acids change the properties of PepT1 in a way that confirms that these parts of the protein indeed act as an electrostatic gate in the transport process. The identification of the structural basis of the functional mechanism of this transporter is important because, in addition to its role in nutrient uptake, PepT1 represents a major pathway for the absorption of several therapeutic drugs.

Topics: Amino Acid Substitution; Animals; Arginine; Aspartic Acid; Cell Membrane; Dipeptides; Electrophysiological Phenomena; Female; Histidine; Hydrogen-Ion Concentration; Ion Channel Gating; Membrane Potentials; Oocytes; Patch-Clamp Techniques; Peptide Transporter 1; Protons; Rabbits; RNA, Complementary; Static Electricity; Symporters; Xenopus laevis

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.

Topics: Amino Acid Substitution; Animals; Biological Transport; Dipeptides; Hydrogen-Ion Concentration; Mutagenesis, Site-Directed; Oocytes; Patch-Clamp Techniques; Peptide Transporter 1; Rabbits; Substrate Specificity; Symporters; Xenopus laevis