Mefenamic acid (MFA) has anti-convulsant and pro-convulsant effects in vivo, and has been shown to potentiate and inhibit GABAA (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 GABAA receptors to resolve the molecular mechanisms by which MFA modulates GABAA receptor function. We demonstrate that MFA potentiated GABA-activated currents for alpha1beta2 gamma2S (EC50 = 3.2 +/- 0.5 microM), but not for alpha1beta1 gamma2S receptors. MFA also enhanced GABA-activated responses and directly activated alpha1beta2/beta3 GABAA receptors, but inhibited responses to GABA on alpha1beta1 constructs (IC50 = 40 +/- 7.2 microM). A comparison of beta1, beta2 and beta3 subunits suggested that the positive modulatory action of MFA involved asparagine (N) 290 in the second transmembrane domain (TM2) of the beta2 and beta3 subunits. Mutation of N290 to serine (S) markedly reduced modulation by MFA in alpha1beta2(N290S)gamma2S receptors, whereas alpha1beta1(S290N)gamma2S constructs revealed potentiated responses to GABA (EC50 = 7.8 +/- 1.7 microM) 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 beta1 and beta3 receptors were susceptible to modulation by MFA. Molecular comparisons of MFA, loreclezole and etomidate, agents which exhibit similar selectivity for GABAA receptors, revealed their ability to adopt similar structural conformations. This study indicates that N290 in TM2 of beta2 and beta3 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.