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.

Temperature effects on the kinetic properties of the rabbit intestinal oligopeptide cotransporter PepT1

“…The reliability of the apparent increase of E act at −120 mV at this pH, compared to the value at pH 6.5, is however doubtful, given the lack of stability in the transport current in these conditions (see Fig. 2a, rightmost panel, and [3,20]). …”

Functional characterization of Atlantic salmon (Salmo salar L.) PepT2 transporters

“…10 . Like other peptide transporters, such as mammalian and fish PepT1 (Renna et al ., 2011a; Vacca et al ., 2019) and rat PepT2 (Chen et al ., 1999), the τ / V curves of both PepT2 appeared bell-shaped ( Fig. 10 A and C ).…”

“…The behaviour is related to the accumulation of substrate inside the cell and to the high affinity of the transporter for the substrate. In this case, the net flux at very high substrate concentration results lower than the flux at the concentration proximal to saturation value (Mertl et al ., 2008; Renna et al ., 2011a; Bosdriesz et al ., 2018).…”

Functional characterization of Atlantic salmon (Salmo salar L.) PepT2 transporters

“…The behaviour is related to the accumulation of substrate inside the cell and to the high affinity of the transporter for the substrate. In this case, the net flux at very high substrate concentration results lower than the flux at the concentration proximal to saturation value (Bosdriesz et al., 2018; Mertl et al., 2008; Renna, Oyadeyi et al., 2011).…”