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Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na(+) ions and 1 H(+) and the counter-transport of 1 K(+) ion, which ensures that extracellular glutamate concentrations are maintained in the submicromolar range. In addition to the coupled ion fluxes, glutamate transport activates an uncoupled anion conductance that does not influence the rate or direction of transport but may have the capacity to influence the excitability of the cell. Free Zn(2+) ions are often co-localized with glutamate in the central nervous system and have the capacity to modulate the dynamics of excitatory neurotransmission. In this study we demonstrate that Zn(2+) ions inhibit the uncoupled anion conductance and also reduce the affinity of L-aspartate for EAAT4. The molecular basis for this effect was investigated using site-directed mutagenesis. Two histidine residues in the extracellular loop between transmembrane domains three and four of EAAT4 appear to confer Zn(2+) inhibition of the anion conductance.

1. The effects of niflumic acid on the substrate-gated currents mediated by the glutamate transporter EAAT4 expressed in Xenopus laevis oocytes were examined using radiolabelled substrate flux measurements and two-electrode voltage clamp techniques. 2. Niflumic acid significantly enhanced the substrate-gated currents in EAAT4, without affecting the affinity of the substrates towards EAAT4. At a concentration of 300 microM, niflumic acid caused a 19 +/- 5 % reduction in L-[(3)H]glutamate uptake and no significant effect on the uptake of DL-[(3)H]aspartate. Thus, enhancement of the substrate-gated currents in EAAT4 does not correlate with the rate of substrate transport and suggests that the niflumic acid-induced currents are not thermodynamically coupled to the transport of substrate. 3. Niflumic acid and arachidonic acid co-applied with substrate to EAAT4-expressing oocytes had similar functional consequences. However, niflumic acid still enhanced the L-glutamate-gated current to the same extent in the presence and absence of a saturating dose of arachidonic acid, which suggests that the sites of action of the two compounds are distinct. 4. The EAAT4-mediated currents for the two substrates, L-glutamate and L-aspartate, were not enhanced equally by addition of the same dose of niflumic acid and the ionic composition of the niflumic acid-induced currents was not the same for the two substrates. Protons carry the L-glutamate-gated niflumic acid-induced current and both protons and chloride ions carry the L-aspartate-gated niflumic acid-induced current. 5. These results show that niflumic acid can be used to probe the functional aspects of EAAT4 and that niflumic acid and other non-steroid anti-inflammatory drugs could be used as the basis for the development of novel modulators of glutamate transporters.

Excitatory amino-acid transporters (EAATs) in the central nervous system maintain extracellular glutamate concentrations below excitotoxic levels and may limit the activation of glutamate receptors. Here we report the cloning of a novel human aspartate/glutamate transporter, EAAT4, which is expressed predominantly in the cerebellum. The transport activity encoded by EAAT4 has high apparent affinity for L-aspartate and L-glutamate, and has a pharmacological profile consistent with previously described cerebellar transport activities. In Xenopus oocytes expressing EAAT4, L-aspartate and L-glutamate elicited a current predominantly carried by chloride ions. This chloride conductance was not blocked by components that block endogenous oocyte chloride channels. Thus EAAT4 combines the re-uptake of neurotransmitter with a mechanism for increasing chloride permeability, both of which could regulate excitatory neurotransmission.