Functional characterization of partial and complete transport cycle of the newly cloned Atlantic salmon (Salmo salar) PepT2 (Slc15a2) di/tripeptide transporter

Intestinal and renal di/tripeptide uptake is driven by H+-dependent cotransporters Slc15a1 (PepT1) and Slc15a2 (PepT2) belonging to SoLute Carrier 15 (Slc15) family. While low-affinity/high-capacity PepT1 transporters have been studied in many teleost fish, data on high-affinity/low-capacity PepT2 is still lacking, except for zebrafish (Danio rerio). The partial and complete transport cycle of the newly cloned Atlantic salmon (Salmo salar) PepT2, heterologously expressed in Xenopus laevis oocytes, was studied as a function of potential and external pH using Two Electrode Voltage Clamp technique. Transient-currents analysis showed H+ role in the first steps of transport cycle: total amount of charges moved (Q) and decay time (τ) vs membrane voltage shifted to more positive potential values when extracellular pH decreased. Transport-currents analysis from Gly-Gln dose-response experiments showed higher apparent substrate affinity (K0.5) for pH 5.5 and 6.5 in proximity of physiological membrane potential. Instead, the maximal relative current (Imax) increased for membrane potential more negative than -50 mV and with the acidic conditions. Proline is a peculiar amino acid for its roles in protein synthesis and structure, metabolism, and nutrition, as well as wound healing, antioxidative reactions, and immune responses. To evaluate the transport specificity of salmon PepT2, the currents were recorded in presence of 1mM of Pro-Gly and Gly-Pro at 6.5 pH. Differently from Salmon PepT1s, in the presence of prolin-containing peptide, PepT2 elicit significant transport associated currents independent of proline position. Like the zebrafish orthologs, salmon PepT2 is a high-affinity/low-capacity transporter with remarkable differences in substrate specificity respect to PepT1s protein.