Blockade of GABAA receptor channels by niflumic acid prevents agonist dissociation

The modulation by the nonsteroidal anti-inflammatory drug niflumic acid (NFA) of the GABAA receptor-mediated currents was studied in acutely isolated cerebellar Purkinje cells using the whole-cell recording and fast drug application system. At concentrations of 3–300 μM NFA potentiated GABA (2 μM)-activated currents, and at concentrations of 1–3 mM NFA blocked these responses. The NFA-induced block was strongly voltage-dependent. Analysis of the voltage dependence of the block suggests that the blocking action of NFA is a result of NFA binding at the site located within GABAA channel pore. The termination of GABA and NFA application was followed by a transient increase of the inward current — “tail” current. These data suggest that NFA acts as a sequential open channel blocker, which prevents dissociation of agonist while the channel is blocked. The tail current develops because, prior to dissociation of agonist, the channels that are in the blocked state must return to the close state via the open state. The tail currents were compared in the presence and absence of gabazine, a competitive antagonist that also allosterically inhibits GABA_A receptors. Application of gabazine only during development of tail current did not change neither amplitude nor time course of this current, while noncompetitive antagonists picrotoxin and penicillin blocked it. Protection of tail current from gabazine block indicates that GABA cannot dissociate from the open-blocked state and the agonist was trapped on the receptor while the channel was open. Trapping was specific for the agonist, because the positive allosteric modulator zolpidem (benzodiazepine site agonist) was able to potentiate the tail current in the absence of GABA in the external solution. Our observations provide a model-independent functional support of the hypothesis that open channel block of GABAA channels by NFA prevents an escape of the agonist from its binding sites.