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 K(M) for GABA transport and the IC50 for ACHC inhibition were similar to the presynaptic transporter. Accumulated [H-3]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. We conclude that GAT-I encodes the ACHC-sensitive GABA transporter subtype which, upon expression in mammalian cells, exhibits properties very similar to those of the native system. Sensitivity to tunicamycin suggests that N-linked glycosylation is important for the functional expression of this membrane protein.