RESUMO
Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking, and synaptic excitability. Both protein kinase C (PKC) and A (PKA) are involved in regulation of γ-aminobutyric acid type A (GABA(A)) receptors through phosphorylation. However, the role of PKA in regulating GABA(A) receptors (GABA(A)-R) following acute ethanol exposure is not known. The present study investigated the role of PKA in the effects of ethanol on GABA(A)-R α1 subunit expression in rat cerebral cortical P2 synaptosomal fractions. Additionally, GABA-related behaviors were examined. Rats were administered ethanol (2.0-3.5 g/kg) or saline and PKC, PKA, and GABA(A)-R α1 subunit levels were measured by western blot analysis. Ethanol (3.5 g/kg) transiently increased GABA(A)-R α1 subunit expression and PKA RIIß subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, lower ethanol doses (2.0 g/kg) had no effect on GABA(A)-R α1 subunit levels, although PKA type II regulatory subunits RIIα and RIIß were increased at 10 and 60 min when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA(A)-R α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA(A)-R α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex (LORR) duration. This effect appears to be mediated in part by GABA(A)-R as increasing PKA activity also increased the duration of muscimol-induced LORR. Overall, these data suggest that PKA mediates ethanol-induced GABA(A)-R expression and contributes to behavioral effects of ethanol involving GABA(A)-R.
RESUMO
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 alpha1beta2/3gamma2 subunits. However, the mechanisms of ethanol-mediated internalization are unknown. This study explored the effect of ethanol on surface expression of GABA(A) alpha1 subunit-containing receptors in cultured cerebral cortical neurons and the role of protein kinase C (PKC) beta, gamma, and epsilon 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 alpha1 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 PKCgamma with alpha1 subunits, whereas PKCbeta/alpha1 association and PKCepsilon/alpha1 colocalization were not altered by ethanol exposure. Selective PKCgamma inhibition by transfection of selective PKCgamma small interfering RNAs blocked ethanol-induced internalization of GABA(A) receptor alpha1 subunits, whereas PKCbeta inhibition using pseudo-PKCbeta had no effect. These findings suggest that ethanol exposure selectively alters PKCgamma translocation to GABA(A) receptors and PKCgamma regulates GABA(A) alpha1 receptor trafficking after ethanol exposure.
