Activation of calcineurin underlies altered trafficking of α2 subunit containing GABA(A) receptors during prolonged epileptiform activity

Fast inhibitory signalling in the mammalian brain is mediated by gamma-aminobutyric acid type A receptors (GABA(A)Rs), which are targets for anti-epileptic therapy such as benzodiazepines. GABA(A)Rs undergo tightly regulated trafficking processes that are essential for maintenance and physiological modulation of inhibitory strength. The trafficking of GABA(A)Rs to and from the membrane is altered during prolonged seizures such as in Status Epilepticus (SE) and has been suggested to contribute to benzodiazepine pharmacoresistance in patients with SE. However, the intracellular signalling mechanisms that cause this modification in GABA(A)R trafficking remain poorly understood. In this study, we investigate the surface stability of GABA(A)Rs during SE utilising the low Mg(2+) model in hippocampal rat neurons. Live-cell imaging of super ecliptic pHluorin (SEP)-tagged α(2) subunit containing GABA(A)Rs during low Mg(2+) conditions reveals that the somatic surface receptor pool undergoes down-regulation dependent on N-methyl-d-aspartate receptor (NMDAR) activity. Analysis of the intracellular Ca(2+) signal during low Mg(2+) using the Ca(2+)-indicator Fluo4 shows that this reduction of surface GABA(A)Rs correlates well with the timeline of intracellular Ca(2+) changes. Furthermore, we show that the activation of the phosphatase calcineurin was required for the decrease in surface GABA(A)Rs in neurons undergoing epileptiform activity. These results indicate that somatic modulation of GABA(A)R trafficking during epileptiform activity in vitro is mediated by calcineurin activation which is linked to changes in intracellular Ca(2+) concentrations. These mechanisms could account for benzodiazepine pharmacoresistance and the maintenance of recurrent seizure activity, and reveal potential novel targets for the treatment of SE. This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’.