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

Abstract

Ethanol produces changes in GABA A receptor trafficking and function that contribute to ethanol dependence symptomatology. Extrasynaptic g-aminobutyric acid A receptors (GABA A -R) mediate inhibitory tonic current and are of particular interest because they are potentiated by physiologically relevant doses of ethanol. Here, we isolate GABA A a4d receptors by western blotting in subsynaptic fractions to investigate protein kinase A (PKA) and protein kinase C (PKC) modulation of ethanol-induced receptor trafficking, while extrasynaptic receptor function is determined by measurement of tonic inhibition and responses evoked by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or PKA/PKC modulators. Ethanol exposure (1 hour) did not alter GABA A a4 receptor abundance, but it increased tonic current amplitude, an effect that was prevented by inhibiting PKA, but not PKC. Direct activation of PKA, but not PKC, increased the abundance and tonic current of extrasynaptic a4d receptors. In contrast, prolonged ethanol exposure (4 hours) reduced a4d receptor abundance as well as tonic current, and this effect was also PKA dependent. Finally, PKC activation by ethanol or phorbol-12,13-dibutyrate (PdBu) had no effect on extrasynaptic a4d subunit abundance or activity. We conclude that ethanol alters extrasynaptic a4d receptor function and expression in cortical neurons in a PKA-dependent manner, but ethanol activation of PKC does not influence these receptors. These results could have clinical relevance for therapeutic strategies to restore normal GABAergic functioning for the treatment of alcohol use disorders.

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

“…However, these studies did not isolate synaptic a4 receptors since d subunits were Rise time (ms) 2.4 6 1.0 3.1 6 1.4 2.8 6 0.9 1.9 6 1.0 2.6 6 0.6 Amplitude (pA) 22.1 6 1.2 23.5 6 1.0 22.8 6 1.9 21.6 6 0.9 22.5 6 1.1 Frequency (Hz) 1.9 6 0.4 1.5 6 0.8 2.1 6 0.6 1.8 6 0.4 2.0 6 0. detected in the P2 fraction. Our current results, combined with recent findings demonstrating a lack of effect of PKC on extrasynaptic GABA A a4d receptors Carlson et al, 2016), support the hypothesis that PKC effects are specific for the synaptic GABA A a4 receptors in cortical neurons and that synaptic and extrasynaptic populations of receptors are subject to different methods of regulation. The similarity of ethanol effects in the synaptic fraction and P2 fraction suggests that the P2 fraction largely consists of synaptic components and that the subsynaptic fraction, although functionally relevant , may not be present in sufficient quantities to confound these results.…”

“…We used a 4-hour in vitro ethanol exposure paradigm that recapitulates changes observed after chronic ethanol consumption and withdrawal (Devaud et al, 1997;Kumar et al, 2003;Carlson et al, 2013Carlson et al, , 2016. It was not surprising that PKA inhibition during 4-hour ethanol exposure had no effect, because we previously found that PKA abundance (Carlson et al, 2013) and activity are not altered by ethanol exposure for 4 hours, despite the effects of ethanol at 1 hour.…”

Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons

“…Since the GABA A α4 subunit is present in both synaptic and extrasynaptic populations of GABA A -Rs, we utilized a biochemical approach previously used for glutamate 32 and GABA A receptors 5,33 to investigate both synaptic and extrasynaptic receptors. We validated this approach for separation of GABA A receptors in cortical cultured neurons by probing for synaptic markers neuroligin 2, gephyrin, postsynaptic density protein-95 and for the GABA A δ subunit that is exclusively localized extrasynaptically (Fig 2) 3 .…”

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).…”