The L-type amino acid transporter 1 (LAT1) is an Na ϩ -independent neutral amino acid transporter subserving the amino acid transport system L. Because of its broad substrate selectivity, system L has been proposed to be responsible for the permeation of amino acid-related drugs through the plasma membrane. To understand the mechanisms of substrate recognition, we have examined the LAT1-mediated transport using a Xenopus laevis oocyte expression system. LAT1-mediated [14 C]phenylalanine uptake was strongly inhibited in a competitive manner by aromatic-amino acid derivatives including L-dopa, ␣-methyldopa, melphalan, triiodothyronine, and thyroxine, whereas phenylalanine methyl ester, N-methyl phenylalanine, dopamine, tyramine, carbidopa, and droxidopa did not inhibit [ 14 C]phenylalanine uptake. Gabapentin, a ␥-amino acid, also exerted a competitive inhibition on LAT1-mediated [14 C]phenylalanine uptake. Although most of the compounds that inhibited LAT1-mediated uptake were able to induce the efflux of [ 14 C]phenylalanine preloaded to the oocytes expressing LAT1 through the obligatory exchange mechanism, melphalan, triiodothyronine, and thyroxine did not induce the significant efflux. Based on the experimental and semiempirical computational analyses, it is proposed that, for an aromatic amino acid to be a LAT1 substrate, it must have a free carboxyl and an amino group. The carbonyl oxygen closer to the amino group needs a computed charge of Ϫ0.55ϳϪ0.56 and must not participate in hydrogen bonding. In addition, the hydrophobic interaction between the substrate side chain and the substrate binding site of LAT1 seems to be crucial for the substrate binding. A substrate, however, becomes a blocker once Connolly accessible areas become large and/or the molecule has a high calculated logP value, such as those for melphalan, triiodothyronine, and thyroxine.System L is an amino acid transporter that transports large neutral amino acids in an Na ϩ -independent manner (Oxender and Christensen, 1963;Christensen, 1990). It is a major route through which living cells take up branched or aromatic amino acids from extracellular fluids. In addition, system L, as a basolateral membrane transport system, plays important roles in the absorption of amino acids through the epithelial cells of the small intestine and renal proximal tubules (Christensen, 1990). System L is also essential in the penetration of amino acids through the blood-brain barrier and the placenta barrier (Christensen, 1990). Because of its broad substrate selectivity, system L is proposed to transport not only naturally occurring amino acids but also amino acid-related compounds such as L-dopa, a therapeutic drug for Parkinsonism; melphalan, an anticancer phenylalanine mustard; triiodothyronine and thyroxine, two thyroid hormones; gabapentin, an anticonvulsant; and S-(1,2-dichlorovinyl)-L-cysteine, a neurotoxic cysteine conjugate (Goldenberg et al., 1979;Christensen, 1990;Lakshmanan et al., 1990;Blondeau et al., 1993;Patel et al., 1993;Su et al., 1995...

Abstract. System L (SL), a basolateral amino acid transporter, transports large neutral amino acids (LNAAs) in a Na + -independent manner. Previously, we identified two isoforms of transporters: L-type amino acid transporter 1 (LAT1) and 2 (LAT2) and revealed their distinct substrate selectivity and transport properties. In this study, to establish more stable human LAT1 (hLAT1) and LAT2 (hLAT2) in vitro assay systems, we established mouse cell lines stably expressing hLAT1 (S2-LAT1) and hLAT2 (S2-LAT2). Real-time quantitative RT-PCR analysis revealed that S2-LAT1 and S2-LAT2 cells express hLAT1 and hLAT2 mRNAs at 20 -1000-fold higher levels than those of endogenous mouse Lat1 and Lat2. S2-LAT1 and S2-LAT2 mediated [14 C]L-leucine transport properties were measured and corresponded to results observed via Xenopus oocytes. Using these cells, the data demonstrate that hLAT1 and hLAT2 exhibit different characters in the acceptance of α-methyl amino acids and amino acid-related compounds with bulky side chains such as thyroid hormones and melphalan. S2-LAT1 and S2-LAT2 cells are expected to facilitate hLAT1 and hLAT2 substrate recognition research and contribute to drug development by providing an efficient assay system to screen for chemical compounds that interact with hLAT1 and hLAT2.