Cloning and Functional Expression of a Human Na 1 and Cl 2 -dependent Neutral and Cationic Amino Acid Transporter B 0 1 *

A Na 1 -dependent neutral and cationic amino acid transport system (B 0 1 ) plays an important role in many cells and tissues; however, the molecular basis for this transport system is still unknown. To identify new transporters, the expressed sequence tag database was queried, and cDNA fragments with sequence similarity to the Na 1 /Cl 2 -dependent neurotransmitter transporter family were identified. Based on these sequences, rapid amplification of cDNA ends of human mammary gland cDNA was used to obtain a cDNA of 4.5 kilobases (kb). The open reading frame encodes a 642-amino acid protein named amino acid transporter B 0 1 . Human ATB 0 1 (hATB 0 1 ) is a novel member of the Na 1 /Cl 2 -dependent neurotransmitter transporter family with the highest sequence similarity to the glycine and proline transporters. Northern blot analysis identified transcripts of ; 4.5 kb and ; 2 kb in the lung. Another tissue survey suggests expression in the trachea, salivary gland, mammary gland, stomach, and pituitary gland. Electrophysiology and radiolabeled amino acid uptake measurements were used to functionally characterize the transporter expressed in Xenopus oocytes. hATB 0 1 was found to transport both neutral and cationic amino acids, with the highest affinity for hydrophobic amino acids and the lowest affinity for proline. Amino acid transport was Na 1 and Cl 2 -dependent and was attenuated in the presence of 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid, a system B 0 1 inhibitor. These characteristics are consistent with system B 0 1 amino acid Experiments— L -[4,5- 3 H]Leucine (136 Ci/mmol), L -[2,3,4,5- 3 H]arginine monohydrochloride (71 Ci/mmol), and L -[G- 3 H]glutamic acid (136 Ci/mmol) (Amersham Pharmacia Biotech) were diluted to a concen- tration of 90 n M and used to assess amino acid uptake. After incubation with 3 H-amino acid in the appropriate Ringer ’ s solution, oocytes were immediately washed four times with the same ice-cold solution, individ-ually solubilized in 1% SDS, and counted by liquid scintillation. Initial time course experiments of L -[ 3 H]leucine transport indicated that uptake was linear from 1–10 min (data not shown); consequently, time points of 2 or 5 min were chosen. For ion dependence experiments, oocytes were initially rinsed three times in Na 1 or Cl 2 -free solution.