Functional characterization of Zn2(+)-sensitive GABA transporter expressed in primary cultures of astrocytes from rat cerebral cortex.

The extracellular levels of gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the mammalian cerebral cortex, are regulated by specific high-affinity Na(+)/Cl(-) dependent transporters (GATs). GAT1 mainly expressed in cerebrocortical neurons is thought to play an important role for clearance of GABA in the extracellular fluid, whereas there is a little information available for pharmacological importance for astrocytic GABA transporters. In the present study, we therefore described the functional characterization of GABA transport in primary cultures of astrocytes from rat cerebral cortex and the identification of GABA transporter subtype(s). GABA transport was Na(+) and Cl(-) dependent and saturable with a Michaelis constant (K(t)) of 9.3+/-2.8 microM. Na(+)- and Cl(-)- activation kinetics revealed that the Na(+)-Cl(-)-to-GABA stoichiometry was 2:1:1 and concentrations of Na(+) and Cl(-) necessary for half-maximal transport (K(0.5)(Na) and K(0.5)(Cl)) were 78+/-28 mM and 9.6+/-2.6 mM, respectively. Na(+)-dependent GABA transport was competitively inhibited by various GABA transport inhibitors, especially GAT2- or GAT3-selective inhibitor. In addition, Zn(2+), which has been reported to be a potent inhibitor of GAT3, was found to have a significantly but partially inhibitory effect on the Na(+)-dependent GABA transport in a concentration-dependent manner. Furthermore, reverse transcription-PCR and Western blot analyses revealed that GAT2 and GAT3 are expressed in primary cultures of astrocytes. These results clearly showed that zinc is a useful reagent for separating GAT3 activity from GAT1- and GAT2-activities in CNS. To our knowledge, the present study represents the first report on the inhibitory effect of zinc on the Na(+)-dependent GABA transport in rat cerebrocortical astrocytes.