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 [ 14 C]Gly‐Sar uptake via PEPT1 and PEPT2 with exceptional high affinity ( K i  = 24 µ m and 3.4 µ m , 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‐[ 3 H]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 µ m 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‐[ 3 H]Pro is a valuable substrate for both mechanistic and structural studies of H + /peptide transporter proteins.