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

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Abstract
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 μm, niflumic acid caused a 19 ± 5 % reduction in l-[3H]glutamate uptake and no significant effect on the uptake of dl-[3H]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.