Identification and functional characterization of a Na+-independent large neutral amino acid transporter, LAT1, in human and rabbit cornea.

Abstract

The objective of this research was to investigate the presence of an Na(+)-independent, large neutral amino acid transporter, LAT1, on rabbit corneal epithelium and human cornea. Freshly excised rabbit corneas were used for transport studies and SIRC (a rabbit corneal cell line) cells for uptake studies. Transport and uptake characteristics of [(3)H]-L-phenylalanine were determined at various concentrations and pH. Inhibition studies were conducted in the presence of other L- and D-amino acids and metabolic inhibitors, such as ouabain and sodium azide, and in the absence of sodium to delineate the mechanism of uptake and transport. Reverse transcription-polymerase chain reaction (RT-PCR) for large neutral amino acid transporter-1 (LAT1) was performed on total RNA from rabbit cornea, SIRC cells, and human cornea. SIRC uptake of L-Phe was found to be saturable, with K(m) of 73 +/- 9 microM, V(max) of 2.0 +/- 0.1 nanomoles/min per milligram protein, and K(d) of 0.44 +/- 0.6 microL/min per milligram protein. Uptake was independent of pH, energy, and Na(+); inhibited by D-Leu, D-Phe, and an L-system-specific inhibitor 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH), but not inhibited by L-Ala and charged amino acids. Transport of L-Phe across rabbit cornea was also saturable (K(m) = 33 +/- 8 microM and V(max) = 0.26 +/- 0.03 nanomoles/min per square centimeter), energy independent, and subject to similar competitive inhibition. LAT1 was identified by RT-PCR in rabbit corneal, SIRC, and human corneal RNA. A Na(+)-independent, facilitative transport system, LAT1, was identified and functionally characterized on rabbit cornea. LAT1 was also identified on human cornea.