Residues R282 and D341 act as electrostatic gates in the proton-dependent oligopeptide transporter PepT1

Elena Bossi, Maria Daniela Renna, Rachele Sangaletti, Francesca D'Antoni, Francesca Cherubino, Gabor Kottra, Antonio Peres

Research output: Contribution to journalArticlepeer-review


Non-technical summary: 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. Abstract The effects of mutations in the charge pair residues Arg282 and Asp341 of the rabbit oligopeptide transporter PepT1 have been studied using electrophysiology in mRNA-injected Xenopus oocytes. Substitution of Arg282 with neutral or negatively charged residues produced a shift towards more positive potentials in the characteristics of charge movement with respect to the wild-type form. Conversely replacement of Asp341 with Arg reduced both pre-steady-state and transport currents and produced a negative shift of the charge movement properties. Both kinds of currents remained pH-sensitive in the mutants. All functional mutants were correctly localized on the cell membrane. Removal of the positive charge of Arg282 produced transporters able to generate conspicuous outward currents whose reversal potential was affected by external pH and by substrate concentration. This suggests that the mutants still translocate protons and substrate as a complex. Charged substrates were accepted by the mutants with the same potency order as the wild-type. The results support the idea that Arg282 and Asp341 play the role of electrostatic gates in the PepT1 transport cycle.

Original languageEnglish
Pages (from-to)495-510
Number of pages16
JournalJournal of Physiology
Issue number3
Publication statusPublished - Feb 2011

ASJC Scopus subject areas

  • Physiology


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