The recent explosion in molecular information has led to the suggestion that amino acid transport across epithelial cells is mediated at least partly by heterodimeric protein complexes (Verrey et al. 1999). To identify which putative transport proteins are involved in system L-like transport at the basolateral membrane of the human intestinal cell line Caco-2, we have investigated the Na⁺-independent component of neutral amino acid transport. Specific PCR primers were designed using the published human sequences of the heavy chain subunits (rBAT and 4F2hc) and the system L-like light chain subunits (LAT1 and LAT2). RT-PCR using Caco-2 mRNA produced bands of the expected sizes for LAT1, LAT2, 4F2hc and rBAT when visualised after separation on a 1 % agarose gel. Comparison of the amplified partial cDNA sequences with published data confirmed that the Caco-2 sequences corresponded to LAT-1 (210-1047 bp, 100 %), LAT-2 (173-569 bp, 100 %), rBAT (41-2106 bp, 100 %) and 4F2hc (76-917 bp, 99% the only predicted change in the corresponding amino acid sequence being G193R). A monoclonal anti-human 4F2hc (CD98) antibody (raised in mouse) reacts with a 125 kDa protein believed to be a heterodimer composed of a glycosylated heavy chain (4F2hc) and a non-glycosylated light chain (Mastroberardino et al. 1998). Immunocytochemical studies using Caco-2 monolayers (passage 102-110, 14-17 days post-seeding) demonstrated 4F2hc-like immuno-reactivity (probed with a FITC-tagged anti-mouse secondary antibody) only at the basolateral membrane. Conversely, an anti-rat NBAT antibody (raised in rabbit) showed NBAT/rBAT-like immunoreactivity (probed using a TRITC-conjugated anti-rabbit secondary antibody) solely at the apical membrane of this cell line. [³H] Tryptophan uptake (1 µM, 0.5 µCi ml^-1, 15 min, 37 °C) was measured across the basolateral surface of Caco-2 cell monolayers in Na⁺-free conditions in the presence or absence of the amino acids L-leucine or L-glutamine (1 µM-10 mM). Surprisingly the ability of these two amino acids to inhibit tryptophan uptake was most similar to that observed in the experiments using a human 4F2hc/ASUR4 (Xenopus LAT1) combination (Mastroberardino et al. 1998), e.g. tryptophan uptake 7.8 ± 0.4 pmol cm^-2 (mean ± S.E.M. (n = 14)) was significantly reduced at 500 µM cold substrate by L-leucine (1.4 ± 0.2 pmol cm^-2, P < 0.001) but not L-glutamine (7.3 ± 1.3 pmol cm^-2, P > 0.05; Student’s t test) with K_i values of 71 µM and 1.8 mM, respectively. In summary, these findings suggest the presence of a system L-type transporter at the basolateral membrane of the Caco-2 cell monolayers. The exact combination of transporter subunits responsible for this transport requires further investigation.

This work was supported by the BBSRC (grant number 13/D09145). L.R.R.M. is supported by a BBSRC Agri-Food Committee Studentship.

Mastroberardino, L., Spindler, B., Pfeiffer, R., Skelly, P.J., Loffings, J., Shoemaker, C.B. & Verrey, F. (1998). Nature 395, 288-291.

Verrey, F., Jack, D.L., Paulson, I.T., Saier, M.H. & Pfeiffer, R. (1999). J. Membr. Biol. 172, 181-192.