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 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 μ m , 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.