Transport of amino acids and GABA analogues via the human proton-coupled amino acid transporter, hPAT1: Characterization of conditions for affinity and transport experiments in Caco-2 cells.

The objective of this study was to investigate transepithelial amino acid transport as a function of Caco-2 cell culture time. Furthermore, the objective was to investigate apical uptake characteristics of hPAT1-mediated transport under various experimental conditions. Apical amino acid uptake and transport studies were conducted in Caco-2 monolayers cultured for 4-28 days. Transepithelial transport of the prototypic hPAT1 (SLC36A1) substrates l-proline and glycine were maximal after 21-28 days in culture. Based on proton-dependency and substrate kinetics the major apical uptake and transport of Gly and Pro in Caco-2 cell monolayers is hPAT1-mediated. The apical uptake of Pro is decreased at apical hyperosmolarity conditions. Furthermore we identified the two GABA-analogues, muscimol and THPO as novel hPAT1 substrates. THPO had an affinity for hPAT1 of 11.3mM, whereas muscimol had one of the highest affinities for hPAT1 (1.7mM) reported. Our findings illustrate the suitability of the Caco-2 model for studying hPAT1-mediated transport. Furthermore, the affinity of THPO and muscimol underlines the possible importance of hPAT1 as a transporter for heterocyclic compounds consisting of a 3-isoxazolol moiety, which has been shown to function as a carboxylic acid bioisostere for substrates of the GABA receptor and transport systems.