GABA uptake and release by a mammalian cell line stably expressing a cloned rat brain GABA transporter.
In order to facilitate study of the neuronal GABA transporter and provide a convenient system for potential drug screening, we have established a CHO cell line, designated 1F9, which stably expresses the cloned GABA transporter from rat brain (GAT-1). 1F9 cells transport GABA at levels approximately 300-fold higher than untransfected CHO cells, and GABA transport in these cells has the following properties: (1) a dependence on sodium and chloride ions; (2) higher sensitivity to neuronal subtype uptake inhibitors (DABA and ACHC) than to glial subtype inhibitors (beta-alanine and THPO); and (3) Km (2.5 microM) and IC50 values for various competitive ligands that are comparable with values determined in synaptosomes and brain slices. Given the fidelity with which the 1F9 cell line expresses these characteristics of the native neuronal GABA transporter, we have used it to further address GABA transporter activity. [3H]GABA uptake by 1F9 cells is inhibited approximately 50% by the chloride transport blockers DIDS and SITS. The GABA receptor agonists muscimol and baclofen also inhibit GABA transport; however, the receptor antagonists bicuculline and phaclofen have no effect. 1F9 cells also show release of [3H]GABA release is calcium independent, and is differentially affected by changes in the ion gradient, as well as by the presence of external substrates and uptake blockers. These experiments indicate that 1F9 cells provide a convenient system for the screening of GABA transport inhibitors.
Division of Biology, California Institute of Technology, Pasadena 91125.
J L
JL
Corey
J
J
Guastella
N
N
Davidson
H A
HA
Lester
D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D018756:GABA Antagonists; D050485:GABA Plasma Membrane Transport Proteins; D017136:Ion Transport; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D027361:Organic Anion Transporters; D051381:Rats; D018079:Receptors, GABA / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D002712:Chlorides; D018756:GABA Antagonists; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; D018079:Receptors, GABA; C493415:Slc6a1 protein, rat; D005680:gamma-Aminobutyric Acid; D012964:Sodium