Ethanol Activation of Protein Kinase A Regulates GABAA Receptor Subunit Expression in the Cerebral Cortex and Contributes to Ethanol-Induced Hypnosis

Abstract

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 (GABAA) receptors through phosphorylation. However, the role of PKA in regulating GABAA receptors (GABAA-R) following acute ethanol exposure is not known. The present study investigated the role of PKA in the effects of ethanol on GABAA-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 GABAA-R α1 subunit levels were measured by western blot analysis. Ethanol (3.5 g/kg) transiently increased GABAA-R α1 subunit expression and PKA RIIβ 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 GABAA-R α1 subunit levels, although PKA type II regulatory subunits RIIα and RIIβ 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 GABAA-R α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABAA-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 GABAA-R as increasing PKA activity also increased the duration of muscimol-induced LORR. Overall, these data suggest that PKA mediates ethanol-induced GABAA-R expression and contributes to behavioral effects of ethanol involving GABAA-R.

Regulation of Extrasynaptic GABAA 4 Receptors by Ethanol-Induced Protein Kinase A, but Not Protein Kinase C Activation in Cultured Rat Cerebral Cortical Neurons

“…This change in surface expression was postulated to be due to an increase in synaptic a4bg2 receptors because GABA A d subunit surface expression remained unchanged. Interestingly, the trafficking changes mediated by PKC are mitigated by concurrent activation of PKA by ethanol both in vivo and in vitro (Kumar et al, 2012;Carlson et al, 2013). Furthermore, we found that mice lacking the PKA regulatory subunit RIIb gene have enhanced vulnerability to reductions in extrasynaptic GABA A a4 subunit trafficking after acute ethanol challenge (Carlson et al, 2014).…”

“…Recent studies by our laboratory have elucidated that protein kinases play a major role in facilitating the regulation of GABA A receptors after ethanol exposure (Kumar et al, 2010;Werner et al, 2011;Kumar et al, 2012). Ethanol has long been known to activate both protein kinase A (PKA) (Dohrman et al, 1996) and protein kinase C (PKC) (Messing et al, 1991), and kinases are important regulators of the behavioral effects of ethanol (Harris et al, 1995;Hodge et al, 1999;Thiele et al, 2000;Choi et al, 2008).…”

“…Receptor adaptations were determined using subcellular fractionation of subsynaptic membrane fractions coupled with western blot analysis (Carlson et al, 2014), while functional changes were measured using wholecell patch clamp analysis of tonic currents. We examined the time-dependent effects of ethanol on extrasynaptic GABA A a4 receptor function and trafficking because time-dependent effects of ethanol were observed on synaptic GABA A receptors as well as ethanol activation of PKC and PKA (Kumar et al, 2012;Carlson et al, 2013).…”

Ethanol Activation of Protein Kinase A Regulates GABAAα1 Receptor Function and Trafficking in Cultured Cerebral Cortical Neurons

“…Although our present results demonstrate modulation of GABA A receptor a1 subunit abundance by PKA in vitro, our previous results suggest the same processes occur in vivo (Kumar et al, 2012). Recently, we found that GABA A a1 levels in rat cerebral cortex were increased following an acute ethanol injection, concurrent with an increase in PKA, whereas PKC was unchanged.…”

“…Additionally, PKA has been shown to directly phosphorylate and modulate GABA A receptor activity (Ives et al, 2002;Brandon et al, 2003). Recently, we found that ethanol doseand time-dependently increased PKA membrane levels in rat cerebral cortex, which corresponded to increased GABA A a1 subunit expression (Kumar et al, 2012). The precise physiological consequences of ethanol regulation of GABAergic inhibition via PKA, however, are unclear.…”