Ethanol Regulation of Synaptic GABAA  4 Receptors Is Prevented by Protein Kinase A Activation

Abstract

Ethanol alters GABA A receptor trafficking and function through activation of protein kinases, and these changes may underlie ethanol dependence and withdrawal. In this study, we used subsynaptic fraction techniques and patch-clamp electrophysiology to investigate the biochemical and functional effects of protein kinase A (PKA) and protein kinase C (PKC) activation by ethanol on synaptic GABA A a4 receptors, a key target of ethanol-induced changes. Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or kinase modulators for 4 hours, a paradigm that recapitulates GABAergic changes found after chronic ethanol exposure in vivo. PKA activation by forskolin or rolipram during ethanol exposure prevented increases in P2 fraction a4 subunit abundance, whereas inhibiting PKA had no effect. Similarly, in the synaptic fraction, activation of PKA by rolipram in the presence of ethanol prevented the increase in synaptic a4 subunit abundance, whereas inhibiting PKA in the presence of ethanol was ineffective. Conversely, PKC inhibition in the presence of ethanol prevented the ethanol-induced increases in synaptic a4 subunit abundance. Finally, we found that either activating PKA or inhibiting PKC in the presence of ethanol prevented the ethanol-induced decrease in GABA miniature inhibitory postsynaptic current decay t 1 , whereas inhibiting PKA had no effect. We conclude that PKA and PKC have opposing effects in the regulation of synaptic a4 receptors, with PKA activation negatively modulating, and PKC activation positively modulating, synaptic a4 subunit abundance and function. These results suggest potential targets for restoring normal GABAergic functioning in the treatment of alcohol use disorders.

Maternal care‐related differences in males and females rats’ sensitivity to ethanol and the associations between the GABAergic system and steroids in males

“…Increased GABA A receptor subunit expressions in the cerebral cortex of Low-LG males, as we observed, suggest a positive relationship between GABA A receptor expression and sensitivity to ethanol-induced hypnosis (Carlson, Bohnsack, & Morrow, 2016; Carlson, Bohnsack, Patel, & Morrow, 2016; Carlson, Kumar, Werner, Comerford, & Morrow, 2013; Kumar et al, 2012; Kumar et al, 2010). The difference in GABA A receptor subunit expression between Low- and High-LG males may be related to differences in circulating steroid levels.…”

Histone deacetylases mediate GABAA receptor expression, physiology, and behavioral maladaptations in rat models of alcohol dependence

“…Previous studies have demonstrated that chronic ethanol exposure causes decreases in GABAergic inhibition in the mPFC [46], hippocampus [4], and amygdala [47] that appear to involve loss of α1 expression. We observed changes in synaptic transmission via decreases in decay-tau that are potentially due to decreased α1 expression and/or increased α4 expression [16,48,49]. Decreases in α1 expression and increases in α4 expression are thought to mediate changes in benzodiazepine sensitivity, seizure susceptibility, and increased anxiety-like behavior [9,14,50,51].…”

Synaptic targets: Chronic alcohol actions

“…Similarly, acute EtOH no longer facilitated GABA- or muscimol-stimulated Cl- uptake in the cortex (Morrow et al, 1988) and cerebellum (Allan and Harris, 1987) after chronic EtOH exposure. More recently, the Morrow laboratory has investigated two distinct populations of synaptic and extrasynaptic α4-containing GABA A Rs in cultured rat cortical neurons (Carlson et al, 2016a; Carlson et al, 2016b). They found that chronic EtOH treatment alters the abundance of both types of subunits, while six weeks of chronic EtOH diet, but not 2 weeks, increased mouse thalamic GABA A R α4 subunit levels (Werner et al, 2016).…”

“…Recently, the Morrow laboratory reported that either PKA activation or PKC inhibition prevented ethanol-induced increases in α4 subunit expression and decreases in the decay of GABA mIPSCs, whereas PKA inhibition had no effect (Bohnsack et al, 2016; Carlson et al, 2016a). This work suggests that PKA and PKC have opposing effects on synaptic α4-containing GABA A Rs, with PKA activation negatively modulating, and PKC activation positively modulating, their abundance and function (Bohnsack et al, 2016; Carlson et al, 2016a). Furthermore, they reported that PKA activation decreases synaptic α4 expression via increased β3 S408/409 phosphorylation, while PKC activation acts via γ2 S327 phosphorylation to increase α4.…”