Functional identification of ASCT1 neutral amino acid transporter as the predominant system for the uptake of L-serine in rat neurons in primary culture.
The uptake of L-serine, a nonessential amino acid known to be transported by the neutral amino acid transporter system ASC, was studied in primary cultures of rat neurons and astrocytes, and compared with that in human embryonic kidney (HEK293) cells transfected with rat ASCT1 cDNA. We first cloned neutral amino acid transporter ASCT1 from rat neurons in primary culture as a transporter candidate for L-serine uptake in the brain. The predicted amino acid sequence from rat ASCT1 exhibited significant homology with mouse and human ASCT1s. The amino acid sequence of rat ASCT1 was 92 and 84% identical to that of mouse and of human ASCT1, respectively. HEK293 cells expressing the rat ASCT1 cDNA showed a saturable dose-dependent and Na(+)-dependent increase in L-[(3)H] serine uptake by high affinity ( K(m) = 67 microM). The substrate selectivity of rat ASCT1 was the same as those of the mouse and human transporter. Northern blot analysis revealed that ASCT1 mRNA was ubiquitously expressed in the brain, with its highest concentration in the striatum and hippocampus. When the uptake of L -[(3)H] serine into rat primary neurons or astrocytes was compared with that of HEK293 cells expressing rat ASCT1 or rat ASCT2 cDNA, the inhibition profile of amino acids for the rat neurons quite resembled that for HEK293 cells expressing rat ASCT1. In contrast, the profile for rat astrocytes was a mixture of that for HEK293 cells expressing rat ASCT1 and that for the cells expressing rat ASCT2. Furthermore, L-[(3)H] serine uptake in neurons was fully Na(+)-dependent. ASCT1 mRNA was expressed in both primary neurons and astrocytes, whereas ASCT2 mRNA was expressed only in astrocytes, as determined by using RT-PCR with primers specific for the rat ASCT1 or rat ASCT2 transporter. Taken together, these findings indicate that ASCT1 predominantly contributes to the uptake of L-serine in primary neurons.
Yamamoto
Toshifumi
T
Laboratory of Molecular Recognition, Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan. yamamoto@yokohama-cu.ac.jp
Nishizaki
Itone
I
Nukada
Toshihide
T
Kamegaya
Etsuko
E
Furuya
Shigeki
S
Hirabayashi
Yoshio
Y
Ikeda
Kazutaka
K
Hata
Harumi
H
Kobayashi
Hideaki
H
Sora
Ichiro
I
Yamamoto
Hideko
H
D000595:Amino Acid Sequence; D026962:Amino Acid Transport System ASC / Q000378:metabolism*; D026905:Amino Acid Transport Systems; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D015152:Blotting, Northern; D001921:Brain / Q000166:cytology*; D002478:Cells, Cultured; D002794:Choline / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods; D004622:Embryo, Mammalian; D006801:Humans; D007668:Kidney; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012694:Serine / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000378:metabolism
D026962:Amino Acid Transport System ASC; D026905:Amino Acid Transport Systems; D015778:Minor Histocompatibility Antigens; D012333:RNA, Messenger; C498848:SLC1A4 protein, human; C420506:SLC1A5 protein, human; C420504:Slc1a4 protein, rat; C498849:Slc1a5 protein, rat; D014316:Tritium; D012965:Sodium Chloride; D012694:Serine; D002794:Choline