Expression of a cloned gamma-aminobutyric acid transporter in mammalian cells

Biochemistry. 1992 Feb 25;31(7):1974-9. doi: 10.1021/bi00122a011.


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)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biological Transport / drug effects
  • Carrier Proteins*
  • Chlorides / metabolism
  • Cloning, Molecular
  • DNA / genetics
  • GABA Plasma Membrane Transport Proteins
  • Gene Expression
  • Genetic Vectors
  • HeLa Cells
  • Humans
  • Kinetics
  • L Cells
  • Membrane Proteins*
  • Membrane Transport Proteins*
  • Mice
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / metabolism
  • Organic Anion Transporters*
  • Plasmids
  • Precipitin Tests
  • Rats
  • Sodium / metabolism
  • Transfection
  • Tunicamycin / pharmacology
  • Xenopus
  • gamma-Aminobutyric Acid / metabolism


  • Carrier Proteins
  • Chlorides
  • GABA Plasma Membrane Transport Proteins
  • Membrane Proteins
  • Membrane Transport Proteins
  • Nerve Tissue Proteins
  • Organic Anion Transporters
  • SLC6A1 protein, human
  • Slc6a1 protein, mouse
  • Slc6a1 protein, rat
  • Tunicamycin
  • gamma-Aminobutyric Acid
  • DNA
  • Sodium