A number of sodium-dependent neutral amino acid transport systems have been functionally characterized to date, including system A, system ASC and system B⁰. The latter was defined from studies in the bovine renal cell line NBL-1, and unlike systems A and ASC binds aromatic amino acids such as phenylalanine, although it transports them poorly (Doyle & McGivan, 1992). An amino acid transporter cloned from the human choriocarcinoma cell line JAR was described as system B⁰ (ATB⁰, Kekuda et al. 1996) although alanine uptake into JAR cells is virtually insensitive to inhibition by phenylalanine (Torres-Zamorano et al. 1997). Here we report the cloning and characterization of a system B⁰ amino acid transporter from NBL-1 cells.

The clone isolated from NBL-1 cells (NBL-1 B⁰, Genbank accession AY039236) encodes a protein of 539 amino acids, which when expressed in Xenopus oocytes induces the transport of alanine (initial rate of control uptake 8.64 ± 0.48, NBL-1 cRNA injected 198 ± 16.6 pmol oocyte h^-1, mean ± S.E.M., n = 5) which is sodium dependent (lithium replacement 71.9 ± 3.02). A 100-fold excess of various amino acids produces the results seen in Table 1, which are characteristic for system B⁰, i.e. neutral, branched and aromatic amino acids are inhibitors. The lack of effect of N-aminoisobutyric acid (MeAIB) shows that this is not system A. In NBL-1 cells, there is no inhibition of alanine uptake by glutamate or aspartate at pH 5.5, indicating that this is not system ASCT2. The inhibition of alanine uptake by phenylalanine in NBL-1 expressing oocytes was shown from a Dixon plot to be competitive (predicted K_i 1.4 mM), although phenylalanine itself is not transported. These findings are in agreement with the original characterization of system B⁰ in NBL-1 cells, and distinguish this transporter from the neutral amino acid transport system found in bovine renal brush-border membranes that mediates the uptake of phenylalanine from the proximal tubule (Lynch & McGivan, 1987). In addition, NBL-1 B⁰ activity is found on the basolateral membrane of the NBL-1 cells (Doyle & McGivan, 1992), which are more like cells from renal distal tubule.

The NBL-1 B⁰ clone has 82.6 % identity at the amino acid level to ASCT2 (system ASC) and 87.8 % identity to JAR B⁰, but there are subtle differences in the kinetics of these transporters as ASCT2 is inhibited by anionic amino acids at low pH, whereas JAR B⁰ is much less sensitive to phenylalanine inhibition. In conclusion, NBL-1 B⁰ is a new member of the superfamily of amino acid transporter genes which includes ASCT2 and ATB⁰, with differences in transporter phenotype brought about by intriguingly small changes in sequence. Phylogenetic comparisons of the sequences of these transporters support this view, with JAR B⁰ and NBL-1 B⁰ having more in common with each other than with ASCT2.

We thank The Wellcome Trust, MRC and Royal Society for funding these studies.