Influence of a proton gradient on the transport kinetics of the H+/amino acid cotransporter PAT1 in Caco-2 cells

The recently cloned proton-coupled amino acid transporter 1 (PAT1) not only accepts several amino acids as substrates but also pharmaceutically relevant l-proline or GABA derivatives such as cis-4-hydroxy-l-proline, l-azetidine-2-carboxylic acid (LACA), 3-amino-1-propanesulfonic acid, nipecotic acid, and the antituberculotic agent d-cycloserine. Because human intestine expresses hPAT1 at the brush border membrane, the transporter may serve as a new oral drug delivery route. Using the human intestinal cell line Caco-2, we have investigated the influence of an inwardly directed proton gradient on the kinetic parameters of l-proline uptake. H+ altered only the apparent affinity of l-proline transport and not the maximal transport velocity. Similarly, treatment of the cells with diethylpyrocarbonate (DEPC), known to chemically modify histidyl residues and block their function, affected only the Kt value of l-proline transport. Both increasing pH and DEPC treatment strongly increased the inhibition constants (Ki) of several drugs at hPAT1. It is concluded that H+ stimulates hPAT1 primarily by increasing the substrate affinity with no detectable influence on the maximal transport velocity of the transporter.