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 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.
Poulsen
M V
MV
Department of Pharmacology D06, University of Sydney, Sydney, NSW 2006, Australia. mvp1975@hotmail.com
Vandenberg
R J
RJ
D027322:Amino Acid Transport System X-AG; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D016718:Arachidonic Acid / Q000494:pharmacology; D001224:Aspartic Acid / Q000494:pharmacology; D004357:Drug Synergism; D004553:Electric Conductivity; D050581:Excitatory Amino Acid Transporter 4; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D009544:Niflumic Acid / Q000494:pharmacology*; D009865:Oocytes; D017470:Receptors, Glutamate / Q000502:physiology*; D013379:Substrate Specificity; D027981:Symporters; D014982:Xenopus laevis
D027322:Amino Acid Transport System X-AG; D000894:Anti-Inflammatory Agents, Non-Steroidal; D050581:Excitatory Amino Acid Transporter 4; D050491:Glutamate Plasma Membrane Transport Proteins; D017470:Receptors, Glutamate; C493391:SLC1A6 protein, human; D027981:Symporters; D016718:Arachidonic Acid; D001224:Aspartic Acid; D018698:Glutamic Acid; D009544:Niflumic Acid