Activation of calcineurin underlies altered trafficking of α2 subunit containing GABAA receptors during prolonged epileptiform activity

Abstract

Fast inhibitory signalling in the mammalian brain is mediated by gamma-aminobutyric acid type A receptors (GABAARs), which are targets for anti-epileptic therapy such as benzodiazepines. GABAARs undergo tightly regulated trafficking processes that are essential for maintenance and physiological modulation of inhibitory strength. The trafficking of GABAARs 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 GABAAR trafficking remain poorly understood. In this study, we investigate the surface stability of GABAARs during SE utilising the low Mg2+ model in hippocampal rat neurons. Live-cell imaging of super ecliptic pHluorin (SEP)-tagged α2 subunit containing GABAARs during low Mg2+ conditions reveals that the somatic surface receptor pool undergoes down-regulation dependent on N-methyl-d-aspartate receptor (NMDAR) activity. Analysis of the intracellular Ca2+ signal during low Mg2+ using the Ca2+-indicator Fluo4 shows that this reduction of surface GABAARs correlates well with the timeline of intracellular Ca2+ changes. Furthermore, we show that the activation of the phosphatase calcineurin was required for the decrease in surface GABAARs in neurons undergoing epileptiform activity. These results indicate that somatic modulation of GABAAR trafficking during epileptiform activity in vitro is mediated by calcineurin activation which is linked to changes in intracellular Ca2+ 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’.

Neuronal activity mediated regulation of glutamate transporter GLT‐1 surface diffusion in rat astrocytes in dissociated and slice cultures

“…Fluorescence was captured using an Olympus microscope (BX51WI) with a 60× water objective (NA: 0.9) Olympus objective coupled to an EM‐CCD camera (Ixon; Andor; Eckel et al, ; Muir et al, ; Muir and Kittler, ; Smith et al, ). Excitation was provided by a mercury‐spiked xenon arc lamp (Cairn) or by a metal‐halide lamp (X‐Cite120, EXFO).…”

Pathophysiology of status epilepticus

“…It has long been recognized that benzodiazepines lose their potency as status epilepticus continues and that this is partly due to the internalization of synaptic (gammasubunit) containing GABA(A) receptors [35][36][37]. This involves the activation of NMDA receptors and then calcineurin-dependent internalization of the GABA(A) receptors [38,39]. This process points to three specific treatment approaches.…”

Status epilepticus: Role for etiology in determining response to benzodiazepines

“…Other studies have demonstrated that phosphatase inhibition can block activity-dependent GABAR trafficking during SE. 11,25 What is not known is whether the dephosphorylation of the b subunit during SE resulted from a promiscuous process that leads to dephosphorylation of all the receptor's phosphorylation sites or a site-directed process that leads to posttranslational changes at a limited number of phosphorylation sites. To confirm and extend that prior finding, 13 the phosphorylation of the b3 (S408/409) and c2 (S327) subunits was determined after LiPilo-SE of 1-hour duration and KA-SE of 3-hour duration.…”