Niflumic acid modulates uncoupled substrate-gated conductances in the human glutamate transporter EAAT4

1. The effects of niflumic acid on the substrate-gated currents mediated by the glutamate transporter EAAT4 expressed in Xenopus laevis oocytes were e xamined using radiolabelled substrate flux measurements and two-electrode v oltage clamp techniques. 2. Niflumic acid significantly enhanced the substrate-gated currents in EAA T4, without affecting the affinity of the substrates towards EAAT4. At a co ncentration of 300 muM, niflumic acid caused a 19 +/- 5% reduction in L-[H- 3]glutamate uptake and no significant effect on the uptake of DL-[H-3]aspar tate. Thus, enhancement of the substrate-gated currents in EAAT4 does not c orrelate with the rate of substrate transport and suggests that the niflumi c acid-induced currents are not thermodynamically coupled to the transport of substrate. 3. Niflumic acid and arachidonic acid co-applied with substrate to EAAT4-ex pressing oocytes had similar functional consequences. However, niflumic aci d still enhanced the L-glutamate-gated current to the same extent in the pr esence 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-as partate, were not enhanced equally by addition of the same dose of niflumic acid and the ionic composition of the niflumic acid-induced currents was n ot the same for the two substrates. Protons carry the L-glutamate-gated nif lumic acid-induced current and both protons and chloride ions carry the L-a spartate-gated niflumic acid-induced current. 5. These results show that niflumic acid can be used to probe the functiona l aspects of EAAT4 and that niflumic acid and other non-steroid anti-inflam matory drugs could be used as the basis for the development of novel modula tors of glutamate transporters.