Molecular mechanisms of drug transport across cell membrane

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One of the remarkable features of the transporters responsible for the transmembrane transport of organic substances is their multispecificity in the substrate recognition, whose molecular mechanisms remain to be clarified. Even after the three-dimensional structures of the transporter proteins are solved by crystallography, it would be still difficult to understand the structural basis for the multispecific nature in the substrate recognition. Therefore, in the present investigation, we have performed structure-activity analysis using molecular pharmacological analysis.Neutral amino acid transporter LAT1 (L-type amino acid transporter 1) transports not only naturally-occurring L-amino acids nut also amino acid related drugs. In order to generate stably transected cell lines, we transected human LAT1 cDNA to S2 cells derived from mouse renal proximal tubule S2 segment of transgenic mice overexpressing SV40 large T antigen. We, then, examined the inhibitory effects of the compounds on the uptake of radiolabeled phenylalanine by the human LAT1-stable transfected cell line. We demonstrated that the phenylalanine uptake was inhibited by aromatic amino acid-related compounds such as L-dopa, alpha-methyldopa, triiodethyronine, thyroxine in a competitive manner. On the other hands phenylalanine-methylester, N-methylphenylalanine and dopamine had no effects on the LAT1-mediated transport. Computational analysis results indicated that presence of aromatic hydrophobic side chains as well as free carboxyl and free amino groups is essential to interacted with substrate binding site of LAT1.It was proved that the combination of the molecular pharmacological analysis and the cpmput ational analysis is the excellent means to reveal the structural basis of the substrate recognition of transporters.