No abstract

Several proline derivatives such as L-azetidine-2-carboxylic acid, cis-4-hydroxy-L-proline, and 3,4-dehydro-DL-proline prevent procollagen from folding into a stable triple-helical conformation, thereby reducing excessive deposition of collagen in fibrotic processes and the growth of tumors. This study was performed to investigate whether the recently discovered human proton-coupled amino acid transporter 1 (hPAT1) is capable of transporting such pharmacologically relevant proline derivatives and also GABA analogs. Uptake of L- 3 H]proline uptake and flux inhibition. We conclude that 1) the substrate specificity of hPAT1 is very much broader than so far reported and 2) the system accepts therapeutically relevant proline and GABA derivatives. hPAT1 is a promising candidate for new ways of oral drug delivery.

The proton-coupled amino acid transporter PAT1, cloned recently from brain and intestine, mediates the uphill transport of l- and d-proline, l-alanine, glycine, taurine, d-serine, GABA, and many other related compounds and drugs. Here we describe the novel finding that l-tryptophan and its derivatives tryptamine, 5-hydroxy-l-tryptophan, serotonin, and indole-3-propionic acid strongly inhibit H+-dependent l-[3H]proline uptake into Caco-2 cells with inhibition constants (K(i)) of 0.9 to 6.1 mM. Uptake of l-[3H]tryptophan into Caco-2 cells on the other hand was not inhibited by l-proline. Whereas PAT1 substrates produced significant changes in a membrane potential assay for electrogenic transport in Caco-2 cells, l-tryptophan, tryptamine, and 5-hydroxy-l-tryptophan failed to alter membrane voltage. When PAT1 was expressed in Xenopus laevis oocytes and analyzed by the two-electrode voltage clamp technique, glycine elicited high inward currents that were dependent on membrane potential but no currents were observed with l-tryptophan, tryptamine, 5-hydroxy-l-tryptophan, or serotonin. Although not transported electrogenically by PAT1, l-tryptophan and its derivatives inhibited glycine-evoked currents dose-dependently. We conclude that serotonin, l-tryptophan, and tryptamine bind to PAT1 with potencies similar to the prototype substrates, inhibit transport function but are not transported by this carrier protein. They may be considered as the carriers' naturally occurring inhibitors that may alter the transport function of PAT1.

The proton coupled amino acid transporter PAT1 expressed in intestine, brain, and other organs accepts L- and D-proline, glycine, and L-alanine but also pharmaceutically active amino acid derivatives such as 3-amino-1-propanesulfonic acid, L-azetidine-2-carboxylic acid, and cis-4-hydroxy-D-proline as substrates. We systematically analyzed the structural requirements for PAT1 substrates by testing 87 amino acids, proline homologs, indoles, and derivatives. Affinity data and effects on membrane potential were determined using Caco-2 cells. For aliphatic amino acids, a blocked carboxyl group, the distance between amino and carboxyl group, and the position of the hydroxyl group are affinity limiting factors. Methylation of the amino group enhances substrate affinity. Hetero atoms in the proline template are well tolerated. Aromatic alpha-amino acids display low affinity. PAT1 interacts strongly with heterocyclic aromatic acids containing an indole scaffold. The structural requirements of PAT1 substrates elucidated in this study will be useful for the development of prodrugs.

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1.