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

Abstract

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 K t value of l-proline transport. Both increasing pH and DEPC treatment strongly increased the inhibition constants ( K i) 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.