Subunit-selective modulation of GABAA receptors by the non-steroidal anti-inflammatory agent, mefenamic acid

Mefenamic acid (MFA) has anti-convulsant and pro-convulsant effects in vivo, and has been shown to potentiate and inhibit GABA(A) (gamma-aminobutyric acid) receptors in vitro. In this study, whole-cell currents were recorded from Xenopus oocytes and human embryonic kidney (HEK) cells expressing human recombinant GABA(A) receptors to resolve the molecular mechanisms by which MFA modulates GABA(A) receptor function. We demonstrate that MFA potentiated GABA-activated currents for alpha 1 beta 2 gamma 2S (EC50 = 3.2 +/- 0.5 mu M), but not for alpha 1 beta 1 gamma 2S receptors. MFA also enhanced GABA-activated responses and directly activated alpha 1 beta 2/beta 3 GABA(A) receptors, but inhibited responses to GABA on alpha 1 beta 1 constructs (IC50 = 40 +/- 7.2 mu M). A comparison of beta 1, beta 2 and beta 3 subunits suggested that the positive modulatory action of MFA involved asparagine (N) 290 in the second transmembrane domain (TM2) of the beta 2 and beta 3 subunits. Mutation of N290 to serine (S) markedly reduced modulation by MFA in alpha 1 beta 2(N290S)gamma 2S receptors, whereas alpha 1 beta 1(S290N)gamma 2S constructs revealed potentiated responses to GABA (EC50 = 7.8 +/- 1.7 mu M) and direct activation by MFA. The potentiation by MFA displayed voltage sensitivity. The direct activation, potentiation and inhibitory aspects of MFA action were predominantly conferred by the beta subunits as the spontaneously active homomeric beta 1 and beta 3 receptors were susceptible to modulation by MFA. Molecular comparisons of MFA, loreclezole and etomidate, agents which exhibit similar selectivity for GABA(A) receptors, revealed their ability to adopt similar structural conformations. This study indicates that N290 in TM2 of beta 2 and beta 3 subunits is important for the regulation of GABAA receptor function by MFA. Our data provide a potential molecular mechanism for the complex central effects of MFA in vivo.