Ethanol Regulation of Synaptic GABAA α4 Receptors Is Prevented by Protein Kinase A Activation

Abstract

Ethanol alters GABAA 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 GABAA α4 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 α4 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 α4 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 α4 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 τ1, whereas inhibiting PKA had no effect. We conclude that PKA and PKC have opposing effects in the regulation of synaptic α4 receptors, with PKA activation negatively modulating, and PKC activation positively modulating, synaptic α4 subunit abundance and function. These results suggest potential targets for restoring normal GABAergic functioning in the treatment of alcohol use disorders.

Fig. 1.

PKA activation prevents ethanol-induced increases in P2 fraction GABA_A α4 subunits. Cortical neurons were exposed to vehicle, ethanol (50 mM), Rp-cAMP (50 μM), forskolin (10 μM), and/or rolipram (10 μM) for 4 hours, followed by P2 fractionation and western blot analysis. (A) Exposure to ethanol for 4 hours increased P2 fraction levels of the GABA_A α4 subunit, which was not affected by coexposure with Rp-cAMP. (B and C) Exposure to the adenylyl cyclase activator forskolin (B) or the phosphodiesterase inhibitor rolipram (C) with ethanol prevented the increase in P2 fraction levels of α4 subunits induced by ethanol. *P < 0.05 (one-way ANOVA, Bonferroni post-test, n = 6–8 per group). Ctrl, control; EtOH, ethanol; Forsk, forskolin; OD, optical density; Roli, rolipram.

Fig. 2.

PKA activation prevents the increased abundance of synaptic α4 receptors induced by ethanol. Cortical neurons were exposed to vehicle, ethanol (50 mM), Rp-cAMP (50 μM), and/or rolipram (10 μM) for 4 hours, followed by synaptic fractionation and western blot analysis. (A) Ethanol decreased synaptic GABA_A α4 abundance, an effect that was not altered by coexposure with Rp-cAMP. (B) Coexposure with rolipram prevented ethanol-induced increases in synaptic α4 abundance. (C and D) GABA_A γ2 abundance was not altered by exposure to ethanol or either Rp-cAMP (C) or rolipram (D). *P < 0.05 (one-way ANOVA, Bonferroni post-test, n = 6–10). Ctrl, control; EtOH, ethanol; OD, optical density; Roli, rolipram.

Fig. 3.

PKC activation by ethanol increases GABA_A α4 subunit levels in the synaptic fraction. Cortical neurons were exposed to vehicle, ethanol (50 mM), CalC (0.3 μM), and/or PdBu (0.1 µM) for 4 hours followed by synaptic fractionation and western blot analysis. (A) Exposure to ethanol for 4 hours increased synaptic fraction levels of the GABA_A α4 subunit, which was prevented by coexposure with CalC. (B) Exposure to PdBu mimicked the effect of ethanol on synaptic GABA_A α4 levels. *P < 0.05 (t test or one-way ANOVA, Bonferroni post-test, n = 5 to 6 per group). Ctrl, control; EtOH, ethanol; OD, optical density; PdBu, phorbol-12,13-dibutyrate; CalC, Calphostin C.

Fig. 4.

PKA activation or PKC inhibition mitigate ethanol-induced alterations in mIPSC responses. Whole-cell patch-clamp recordings of cortical neurons were made in the presence of TTX (1 μM), CNQX (10 μM), and AP-5 (40 μM) to isolate GABA mIPSCs after 4-hour exposure to ethanol and/or kinase modulatory drugs. (A) Decay time (decay τ₁) was significantly decreased by ethanol exposure, an affect that was not affected by coexposure of ethanol with Rp-cAMP; however, coexposure of ethanol with rolipram prevented this decrease. (B) Coexposure of ethanol with CalC also prevented the decrease in decay rate induced by ethanol alone. (C) Quantification of changes in decay τ₁ is shown. (*P < 0.05 compared with control, EtOH plus CalC, or EtOH plus rolipram groups; one-way ANOVA, Bonferroni post-test). Summarized mIPSC metrics are presented in Table 1. AP-5, d-2-amino-5-phosphonopentanoic acid; CNQX, 6-cyno-7-nitroquinoxaline-2,3-dione; Ctrl, control; EtOH, ethanol; Roli, rolipram; TTX, tetrodotoxin.