Expression of the GLT-1 subtype of Na+-dependent glutamate transporter: pharmacological characterization and lack of regulation by protein kinase C.
Several subtypes of Na+-dependent glutamate transporters have been pharmacologically differentiated in brain tissues. Five distinct cDNA clones that express Na+-dependent glutamate transport activity have been isolated. One goal of the current study was to compare the pharmacological properties of the rat GLT-1 subtype of transporter to those identified previously using rat brain tissues. To accomplish this goal, GLT-1 was stably transfected into two different cell lines that express low levels of endogenous transport activity (MCB and L-M (TK-)). Several clones stably transfected with GLT-1 were isolated. In each cell line, Na+-dependent glutamate transport activity was saturable with similar Km values (19 and 37 microM). The pharmacological properties of GLT-1-mediated transport in these cell lines paralleled those observed for the predominant pharmacology observed in cortical crude synaptosomes. These data are consistent with other lines of evidence that suggest that GLT-1 may be sufficient to explain most of the Na+-dependent glutamate transport activity in cortical synaptosomes. Although recent studies using HeLa cells have suggested that GLT-1 can be rapidly up-regulated by activation of protein kinase C (PKC), modulation of PKC or phosphatase activity had no effect on GLT-1-mediated activity in these transfected cell lines. To determine if GLT-1 regulation by PKC is cell-specific, HeLa cells, which endogenously express the EAAC1 subtype of transporter, were stably transfected with GLT-1. Although EAAC1-mediated activity was increased by activation of PKC, we found no evidence for regulation of GLT-1. Despite the present findings, GLT-1 activity may be regulated by PKC under certain conditions.
Tan
J
J
Department of Pediatrics and Pharmacology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Zelenaia
O
O
Correale
D
D
Rothstein
J D
JD
Robinson
M B
MB
D018528:ATP-Binding Cassette Transporters / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D007739:L Cells; D051379:Mice; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D012044:Regression Analysis; D013574:Synaptosomes / Q000378:metabolism; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D011994:Recombinant Proteins; D018698:Glutamic Acid; D011493:Protein Kinase C