Expression of a cloned gamma-aminobutyric acid transporter in mammalian cells.
The cDNA clone GAT-1, which encodes a Na(+)- and Cl(-)-coupled GABA transporter from rat brain, has been expressed in mammalian cells using three different systems: (1) transient expression upon transfection of mouse Ltk- cells with a eukaryotic expression vector containing GAT-1; (2) stable expression in L-cells transfected with the same vector; (3) transfection of HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase. Similar results both qualitatively and quantitatively were obtained with all systems. The GABA transporter expressed in HeLa and L-cells retains all the properties described previously for GABA transport into synaptosomes and synaptic plasma membrane vesicles. It was fully inhibited by cis-3-aminocyclohexanecarboxylic acid (ACHC) and not by beta-alanine. The KM for GABA transport and the IC50 for ACHC inhibition were similar to the presynaptic transporter. Accumulated [3H]GABA was released from transfected cells by dissipating the transmembrane Na+ gradient with nigericin or by exchange with unlabeled external GABA. Accumulation was stimulated by both Na+ and Cl- in the external medium. However, in the absence of external Cl-, a small amount of GABA transport remained which was dependent on GAT-1 transfection. Functional expression of the GABA transporter was abolished by tunicamycin. An antitransporter antibody specifically immunoprecipitates a polypeptide with an apparent molecular mass of about 70 kDa from GAT-1-transfected cells. When cells were grown in the presence of tunicamycin, only a faint band of apparent mass of about 60 kDa was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Keynan
S
S
Department of Biochemistry, Hadassah Medical School, Hebrew University of Jerusalem, Israel.
Suh
Y J
YJ
Kanner
B I
BI
Rudnick
G
G
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins; D002712:Chlorides / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D050485:GABA Plasma Membrane Transport Proteins; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D007739:L Cells; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D027361:Organic Anion Transporters; D010957:Plasmids; D011233:Precipitin Tests; D051381:Rats; D012964:Sodium / Q000378:metabolism; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D014981:Xenopus; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; C493413:SLC6A1 protein, human; C493414:Slc6a1 protein, mouse; C493415:Slc6a1 protein, rat; D014415:Tunicamycin; D005680:gamma-Aminobutyric Acid; D004247:DNA; D012964:Sodium