Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234

The availability of amino acids in the brain is regulated by the blood-brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low K-m) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site-directed mutagenesis of the BBB LAT1. The rabbit has a high K-m LAT1 large neutral amino acid transporter, as compared to the low K-m neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the K-m and V-max of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the K-m by 64% and the V-max by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.