Transport properties of a system y+L neutral and basic amino acid transporter. Insights into the mechanisms of substrate recognition.
The properties of system y(+)L-mediated transport were investigated on rat system y(+)L transporter, ry(+)LAT1, coexpressed with the heavy chain of cell surface antigen 4F2 in Xenopus oocytes. ry(+)LAT1-mediated transport of basic amino acids was Na(+)-independent, whereas that of neutral amino acids, although not completely, was dependent on Na(+), as is typical of system y(+)L-mediated transport. In the absence of Na(+), lowering of pH increased leucine transport, without affecting lysine transport. Therefore, it is proposed that H(+), besides Na(+) and Li(+), is capable of supporting neutral amino acid transport. Na(+) and H(+) augmented leucine transport by decreasing the apparent K(m) values, without affecting the V(max) values. We demonstrate that although ry(+)LAT1-mediated transport of [(14)C]l-leucine was accompanied by the cotransport of (22)Na(+), that of [(14)C]l-lysine was not. The Na(+) to leucine coupling ratio was determined to be 1:1 in the presence of high concentrations of Na(+). ry(+)LAT1-mediated leucine transport, but not lysine transport, induced intracellular acidification in Chinese hamster ovary cells coexpressing ry(+)LAT1 and 4F2 heavy chain in the absence of Na(+), but not in the presence of physiological concentrations of Na(+), indicating that cotransport of H(+) with leucine occurred in the absence of Na(+). Therefore, for the substrate recognition by ry(+)LAT1, the positive charge on basic amino acid side chains or that conferred by inorganic monovalent cations such as Na(+) and H(+), which are cotransported with neutral amino acids, is presumed to be required. We further demonstrate that ry(+)LAT1, due to its peculiar cation dependence, mediates a heteroexchange, wherein the influx of substrate amino acids is accompanied by the efflux of basic amino acids.
Kanai
Y
Y
Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan. ykanai@kyorin-u.ac.jp
Fukasawa
Y
Y
Cha
S H
SH
Segawa
H
H
Chairoungdua
A
A
Kim
D K
DK
Matsuo
H
H
Kim
J Y
JY
Miyamoto
K
K
Takeda
E
E
Endou
H
H
D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D001665:Binding Sites; D001692:Biological Transport; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D005452:Fluoresceins; D027261:Fusion Regulatory Protein-1; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D013379:Substrate Specificity; D014981:Xenopus
D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D015703:Antigens, CD; D002352:Carrier Proteins; D005452:Fluoresceins; D027261:Fusion Regulatory Protein-1; C081009:Slc3a1 protein, rat; D012964:Sodium; D007930:Leucine; D008239:Lysine