The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABA_Areceptors (GABA_A-Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to “tonic” inhibition that can control levels of excitability and network activity. The GABA_A-R α4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABA_A-R-mediated tonic inhibition. Mice were engineered to lack the α4 subunit by targeted disruption of theGabra4gene. α4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABA_A-Rs containing the α4 subunit and that gaboxadol achieves its effects via the activation of this GABA_A-R subtype.

The time-dependent effects of ethanol (EtOH) intoxication on GABA_Areceptor (GABA_AR) composition and function were studied in rats. A cross-linking assay and Western blot analysis of microdissected CA1 area of hippocampal slices obtained 1 h after EtOH intoxication (5 g/kg, gavage), revealed decreases in the cell-surface fraction of α4 and δ, but not α1, α5, or γ2 GABA_AR subunits, without changes in their total content. This was accompanied (in CA1 neuron recordings) by decreased magnitude of the picrotoxin-sensitive tonic current (I_tonic), but not miniature IPSCs (mIPSCs), and by reduced enhancement ofI_tonicby EtOH, but not by diazepam. By 48 h after EtOH dosing, cell-surface α4 (80%) and γ2 (82%) subunit content increased, and cell-surface α1 (−50%) and δ (−79%) and overall content were decreased. This was paralleled by faster decay of mIPSCs, decreased diazepam enhancement of both mIPSCs andI_tonic, and paradoxically increased mIPSC responsiveness to EtOH (10–100 mm). Sensitivity to isoflurane- or diazepam-induced loss of righting reflex was decreased at 12 and 24 h after EtOH intoxication, respectively, suggesting functional GABA_AR tolerance. The plastic GABA_AR changes were gradually and fully reversible by 2 weeks after single EtOH dosing, but unexplainably persisted long after withdrawal from chronic intermittent ethanol treatment, which leads to signs of alcohol dependence. Our data suggest that early tolerance to EtOH may result from excessive activation and subsequent internalization of α4βδ extrasynaptic GABA_ARs. This leads to transcriptionally regulated increases in α4 and γ2 and decreases in α1 subunits, with preferential insertion of the newly formed α4βγ2 GABA_ARs at synapses.

Prolonged ethanol exposure causes central nervous system hyperexcitability that involves a loss of GABAergic inhibition. We previously demonstrated that long-term ethanol exposure enhances the internalization of synaptic GABA_A receptors composed of α1β2/3γ2 subunits. However, the mechanisms of ethanol-mediated internalization are unknown. This study explored the effect of ethanol on surface expression of GABA_A α1 subunit-containing receptors in cultured cerebral cortical neurons and the role of protein kinase C (PKC) β, γ, and ε isoforms in their trafficking. Cultured neurons were prepared from rat pups on postnatal day 1 and maintained for 18 days. Cells were exposed to ethanol, and surface receptors were isolated by biotinylation and P2 fractionation, whereas functional analysis was conducted by whole-cell patch-clamp recording of GABA- and zolpidem-evoked responses. Ethanol exposure for 4 h decreased biotinylated surface expression of GABA_A receptor α1 subunits and reduced zolpidem (100 nM) enhancement of GABA-evoked currents. The PKC activator phorbol-12,13-dibutyrate mimicked the effect of ethanol, and the selective PKC inhibitor calphostin C prevented ethanol-induced internalization of these receptors. Ethanol exposure for 4 h also increased the colocalization and coimmunoprecipitation of PKCγ with α1 subunits, whereas PKCβ/α1 association and PKCε/α1 colocalization were not altered by ethanol exposure. Selective PKCγ inhibition by transfection of selective PKCγ small interfering RNAs blocked ethanol-induced internalization of GABA_A receptor α1 subunits, whereas PKCβ inhibition using pseudo-PKCβ had no effect. These findings suggest that ethanol exposure selectively alters PKCγ translocation to GABA_A receptors and PKCγ regulates GABA_A α1 receptor trafficking after ethanol exposure.

J. Neurochem. (2011) 116, 554–563. Abstract Ethanol exposure produces alterations in GABA_A receptor function and expression associated with CNS hyperexcitability, but the mechanisms of these effects are unknown. Ethanol is known to increase both GABA_A receptor α4 subunits and protein kinase C (PKC) isozymes in vivo and in vitro. Here, we investigated ethanol regulation of GABA_A receptor α4 subunit expression in cultured cortical neurons to delineate the role of PKC. Cultured neurons were prepared from rat pups on postnatal day 0–1 and tested after 18 days. GABA_A receptor α4 subunit surface expression was assessed using P2 fractionation and surface biotinylation following ethanol exposure for 4 h. Miniature inhibitory post‐synaptic currents were measured using whole cell patch clamp recordings. Ethanol increased GABA_A receptor α4 subunit expression in both the P2 and biotinylated fractions, while reducing the decay time constant in miniature inhibitory post‐synaptic currents, with no effect on γ2 or δ subunits. PKC activation mimicked ethanol effects, while the PKC inhibitor calphostin C prevented ethanol‐induced increases in GABA_A receptor α4 subunit expression. PKCγ siRNA knockdown prevented ethanol‐induced increases in GABA_A receptor α4 subunit expression, but inhibition of the PKCβ isoform with PKCβ pseudosubstrate had no effect. We conclude that PKCγ regulates ethanol‐induced alterations in α4‐containing GABA_A receptors.