Structural selectivity and molecular nature of L-glutamate transport in cultured human fibroblasts.
Uptake of L-[3H]glutamate by monolayers of fibroblasts cultured from human embryonic skin has been studied in the presence of several nonradioactive structural analogs of glutamate and aspartate. Results have suggested that the structural specificites of glutamate transporters in cultured human fibroblasts are similar to those of glutamate transporters in the mammalian brain. Only subtle differences have been detected: in the mammalian cerebral cortex, enantiomers of threo-3-hydroxyaspartate are almost equipotent as inhibitors of L-[3H]glutamate uptake while, in human fibroblasts, the D-isomer has been found to be an order of magnitude less potent than the corresponding L-isomer. Kinetic analysis of a model in which substrates are recognized by the glutamate transporter binding site(s) as both alpha- and beta-amino acids indicated that such a mechanism cannot explain the apparent negative cooperativity characterizing the effects of D- and L-aspartate. Molecular modeling has been used to estimate the optimum conformation of L-glutamate as it interacts with the transporter(s). Flow cytometry has indicated that all fibroblasts in culture express at least moderate levels of four glutamate transporters cloned from human brain. Small subpopulations (< 3%) of cells, however, were strongly labeled with antibodies against EAAT1 (GLAST) and EAAT2 (GLT-1) transporters. We conclude that these two transporters--known to be strongly expressed in brain tissue--can be principally responsible for the "high affinity" transport of glutamate also in nonneural cells.
Cooper
B
B
Department of Anatomy and Histology, University of Sydney, NSW, Australia.
Chebib
M
M
Shen
J
J
King
N J
NJ
Darvey
I G
IG
Kuchel
P W
PW
Rothstein
J D
JD
Balcar
V J
VJ
D018528:ATP-Binding Cassette Transporters / Q000276:immunology / Q000378:metabolism; D027322:Amino Acid Transport System X-AG; D000906:Antibodies / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D008956:Models, Chemical; D008958:Models, Molecular; D017470:Receptors, Glutamate / Q000276:immunology / Q000378:metabolism; D017981:Receptors, Neurotransmitter / Q000276:immunology / Q000378:metabolism; D012867:Skin / Q000196:embryology / Q000378:metabolism*; D027981:Symporters
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D000906:Antibodies; D002352:Carrier Proteins; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050491:Glutamate Plasma Membrane Transport Proteins; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; C493397:SLC1A2 protein, human; C493394:SLC1A3 protein, human; D027981:Symporters; D001224:Aspartic Acid; D018698:Glutamic Acid