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. 2001 Jul 1;534(Pt 1):159-67.
doi: 10.1111/j.1469-7793.2001.00159.x.

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

M V Poulsen  1 , R J Vandenberg
Affiliations

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

M V Poulsen et al. J Physiol. .

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 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.

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Figures

Figure 1
Figure 1. Niflumic acid enhances the substrate-gated conductances of EAAT4
Representative current traces showing the effects of substrates and niflumic acid on oocytes expressing EAAT4 (A and B) and uninjected oocytes (C), voltage clamped at −60 mV. A, 10 μml-Glu (□), 300 μm niflumic acid (▪). B, 10 μml-Asp (□), 300 μm niflumic acid (▪). Traces have been selected from cells in which niflumic acid gave no response on it own (see text).
Figure 2
Figure 2. The niflumic acid-enhanced current is mediated by EAAT4
Representative current trace from an oocyte expressing EAAT4, voltage clamped at −60 mV. TBOA blocks part of both the l-Asp-induced current and the l-Asp-gated niflumic acid-induced current. ▪, 10 μml-Asp; formula image, 300 μm niflumic acid; □, 300 μm TBOA.
Figure 3
Figure 3. Dose-response curve of l-Glu- and l-Asp-elicited currents at −60 mV in the absence (•) and presence (^) of 300 μm niflumic acid
Points represent means ±s.e.m. from 4 cells for l-Glu (A) and 6 cells for l-Asp (B). The current measurements have been normalized to the maximal current generated by l-Glu and l-Asp in the absence of niflumic acid. Curves represent data fitted to the modified Michaelis-Menten equation (see Methods).
Figure 4
Figure 4. Dose-response relationship for niflumic acid stimulation of l-Asp-elicited currents at −60 mV
l-Asp (10 μm) was co-applied with increasing doses of niflumic acid; as saturation was not achieved at 1 mm niflumic acid, kinetic constants were not calculated. Data represent normalized mean current ±s.e.m. from 7 cells.
Figure 5
Figure 5. The effects of niflumic acid on the uptake of radiolabelled substrate
Uptake of l-[3H]Glu (□) and dl-[3H]Asp (▪). Uninjected oocytes and oocytes expressing EAAT4 were incubated at room temperature in ND96 buffer containing 3H-labelled substrate with or without 300 μm niflumic acid for 10 min. The uptake of l-[3H]Glu was reduced by 19 ± 5 % in the presence of niflumic acid and there was no significant effect on dl-[3H]Asp uptake. Since different batches of oocytes with different expression levels were used, the data were normalized to the uptake in EAAT4 without niflumic acid. Data represent mean uptake ±s.e.m. from 5 cells for each condition.
Figure 6
Figure 6. The effects of niflumic acid and arachidonic acid on EAAT4 are additive
A, after application of 10 μml-Glu to oocytes expressing EAAT4, various doses of arachidonic acid were co-applied. Arachidonic acid did not generate a current when applied to oocytes in the absence of l-Glu. Arachidonic acid caused a dose-dependent enhancement of the l-Glu-gated conductance, with an EC50 of 20 ± 4 μm. Data were fitted to the modified Michaelis-Menten equation and represent mean values ±s.e.m. from 4 cells. B, current traces from a representative cell showing the additive effect of arachidonic acid and niflumic acid on the substrate-gated current from oocytes expressing EAAT4 and voltage clamped at −60 mV. Control responses to l-Glu alone and l-Glu with niflumic acid were measured first. Arachidonic acid was then applied with l-Glu followed by the addition of niflumic acid. After washout of arachidonic acid and niflumic acid, re-application of l-Glu with niflumic acid generated responses similar to control responses. ▪, 10 μml-Glu; □, 100 μm arachidonic acid; formula image, 300 μm niflumic acid. C, mean ±s.e.m. values from 4 cells corresponding to the current traces in B. In the oocytes used for this experiment, niflumic acid (NFA) did not generate a current when applied alone to oocytes expressing EAAT4. AA, arachidonic acid.
Figure 7
Figure 7. The effect of pH on the reversal potential of the niflumic acid-induced currents in EAAT4
All I-V relationships are niflumic acid (NFA)-induced currents: (Isubstrate + NFA + buffer − INFA + buffer) − (Isubstrate + buffer − Ibuffer). A, representative I-V relationships showing the shifts in reversal potential of the l-Glu-gated niflumic acid-induced conductance with pH. B and C, representative I-V relationships showing the l-Asp-gated niflumic acid-induced conductance at different pH values. In B, the extracellular chloride concentration is 104 mm and in C the extracellular chloride concentration is 30 mm. For A-C:•, conductance measured at pH 6.5; ▴, conductance measured at pH 7.5; □, conductance measured at pH 8.5). The reversal potential of the l-Glu-gated niflumic acid-induced current shifted −62 ± 5 mV per 10-fold shift in the H+ concentration (D) whereas for the l-Asp-gated niflumic acid-induced current the reversal potential shifted −29 ± 3 mV with 104 mm Cl− (□) and −33 ± 5 mV with 30 mm Cl− (•) per 10-fold change in the H+ concentration (E).
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