Synthesis and characterization of a new and radiolabeled high-affinity substrate for H⁺/peptide cotransporters

In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H⁺/peptide cotransporters. l-4,4′-Biphenylalanyl- l-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H⁺/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H ⁺/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H⁺/peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the [¹⁴C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (K_i = 24 μm and 3.4 μm, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[³H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and δ-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis-Menten constant (K_t) of 7.6 μm and a maximal velocity (V_max) of 1.1 nmol·30 min^-1·mg of protein^-1. Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[³H]Pro is a valuable substrate for both mechanistic and structural studies of H⁺/peptide transporter proteins.