Subunit compensation and plasticity of synaptic GABA A receptors induced by ethanol in α4 subunit knockout mice

Abstract

There is considerable evidence that ethanol (EtOH) potentiates y-aminobutyric acid type A receptor (GABA AR) action, but only GABA ARs containing δ subunits appear sensitive to low millimolar EtOH. The α4 and δ subunits co-assemble into GABA ARs which are relatively highly expressed at extrasynaptic locations in the dentate gyrus where they mediate tonic inhibition. We previously demonstrated reversible- and time-dependent changes in GABA AR function and subunit composition in rats after single-dose EtOH intoxication. We concluded that early tolerance to EtOH occurs by over-activation and subsequent internalization of EtOH-sensitive extrasynaptic α4βδ-GABA ARs. Based on this hypothesis, any highly EtOH-sensitive GABA ARs should be subject to internalization following exposure to suitably high EtOH doses. To test this, we studied the GABA ARs in mice with a global deletion of the α4 subunit (KO). The dentate granule cells of these mice exhibited greatly reduced tonic currents and greatly reduced potentiation by acutely applied EtOH, whereas synaptic currents showed heightened sensitivity to low EtOH concentrations. The hippocampus of naive KO mice showed reduced δ subunit protein levels, but increased α2, and γ2 levels compared to wild-type (WT) controls, suggesting at least partial compensation by these subunits in synaptic, highly EtOH-sensitive GABA ARs of KO mice. In WT mice, cross-linking and Western blot analysis at 1 h after an EtOH challenge (3.5g/kg, i.p.) revealed increased intracellular fraction of the α1, α4, and δ, but not α2, α5, or γ2 subunits. By contrast, we observed significant internalization of α1, α2, δ, and γ2 sub-units after a similar EtOH challenge in KO mice. Synaptic currents from naïve KO mice were more sensitive to potentiation by zolpidem (0.3μM, requiring α1/α2, inactive at α4/5 GABA ARs) than those from naïve WT mice. At 1 h after EtOH, synaptic currents of WT mice were unchanged, whereas those of KO mice were significantly less sensitive to zolpidem, suggesting decreases in functional α1/2βγ GABA ARs. These data further support our hypothesis that EtOH intoxication induces GABAAR plasticity via internalization of highly EtOH-sensitive GABA ARs. © 2011 Suryanarayanan, Liang, Meyer, Lindemeyer, Chandra, Homanics, Sieghart, Olsen and Spigel-man.