Subunit-selective modulation of GABA(A) 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 huma n recombinant GABA(A) receptors to resolve the molecular mechanisms by whic h MFA modulates GABA(A) receptor function. We demonstrate that MFA potentia ted 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 GA BA-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 (I C50 = 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 sub units. 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)gamm a 2S constructs revealed potentiated responses to GABA (EC50 = 7.8 +/- 1.7 mu M) and direct activation by MFA. The potentiation by MFA displayed volta ge sensitivity. The direct activation, potentiation and inhibitory aspects of MFA action were predominantly conferred by the beta subunits as the spon taneously 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 t heir ability to adopt similar structural conformations. This study indicate s that N290 in TM2 of beta 2 and beta 3 subunits is important for the regul ation of GABAA receptor function by MFA. Our data provide a potential molec ular mechanism for the complex central effects of MFA in vivo.