The mechanisms of potentiation and inhibition of GABAA receptors by non-steroidal anti-inflammatory drugs, mefenamic and niflumic acids

Fenamates mefanamic and niflumic acids (MFA and NFA) induced dual potentiating and inhibitory effects on GABA currents recorded in isolated cerebellar Purkinje cells using the whole-cell patch-clamp and fast-application techniques. Regardless of the concentration, both drugs induced a pronounced prolongation of the current response. We demonstrated that the same concentration of drugs can produce both potentiating and inhibitory effects, depending on the GABA concentration, which indicates that both processes take place simultaneously and the net effect depends on the concentrations of both the agonist and fenamate. We found that the NFA-induced block is strongly voltage-dependent. The Woodhull analysis of the block suggests that NFA has two binding sites in the pore - shallow and deep. We built a homology model of the open GABA(A)R based on the cryo-EM structure of the open alpha 1 GlyR and applied Monte-Carlo energy minimization to optimize the ligand-receptor complexes. A systematic search for MFA/NFA binding sites in the GABA(A)R pore revealed the existence of two sites, the location of which coincides well with predictions of the Woodhull model. In silico docking suggests that two fenamate molecules are necessary to occlude the pore. We showed that MFA, acting as a PAM, competes with an intravenous anesthetic etomidate for a common binding site. We built structural models of MFA and NFA binding at the transmembrane beta( + )/alpha( - ) intersubunit interface. We suggested a hypothesis on the molecular mechanism underlying the prolongation of the receptor lifetime in open state after MFA/NFA binding and beta subunit specificity of the fenamate potentiation.