PKCγ is required for ethanol-induced increases in GABA(A) receptor α4 subunit expression in cultured cerebral cortical neurons

Abstract

Ethanol exposure produces alterations in GABA(A) receptor function and expression associated with CNS hyperexcitability, but the mechanisms of these effects are unknown. Ethanol is known to increase both GABA(A) receptor α4 subunits and protein kinase C (PKC) isozymes in vivo and in vitro. Here, we investigated ethanol regulation of GABA(A) receptor α4 subunit expression in cultured cortical neurons to delineate the role of PKC. Cultured neurons were prepared from rat pups on postnatal day 0-1 and tested after 18 days. GABA(A) receptor α4 subunit surface expression was assessed using P2 fractionation and surface biotinylation following ethanol exposure for 4 h. Miniature inhibitory post-synaptic currents were measured using whole cell patch clamp recordings. Ethanol increased GABA(A) receptor α4 subunit expression in both the P2 and biotinylated fractions, while reducing the decay time constant in miniature inhibitory post-synaptic currents, with no effect on γ2 or δ subunits. PKC activation mimicked ethanol effects, while the PKC inhibitor calphostin C prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression. PKCγ siRNA knockdown prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression, but inhibition of the PKCβ isoform with PKCβ pseudosubstrate had no effect. We conclude that PKCγ regulates ethanol-induced alterations in α4-containing GABA(A) receptors.

Figure 1

Ethanol exposure alters GABA_A receptor α4 subunit expression in cultured cortical neurons. Neurons were exposed to ethanol (50mM) for 4 hours followed by preparation of P2 fractions as well as biotin-labeling of surface proteins followed by western blotting. Representative Western blots are shown for α4 subunit from P2 fractions (A), biotin-labeled surface proteins (B), or cytosolic fractions (C). Data represent mean ± S.E.M., Data were analyzed using Student's t-test. *, (A) t = 6.534, (B) t = 2.426, *p < 0.05 compared to control, n=3-6/group.

Figure 2

Effects of ethanol exposure on GABA_A receptor γ2 and δ subunits. Neurons were exposed to ethanol (50mM) for 4 hours followed by preparation of P2 fractions. Representative western blots are shown for γ2 (A) and δ subunits (B). A. Ethanol exposure did not alter γ2 subunit expression. B. Ethanol exposure did not alter δ subunit expression, but a trend towards a decrease was observed (p < 0.07). Data represent mean ± S.E.M., Student's t-test. n = 4-5 per group.

Figure 3

Representative trace showing the effect of ethanol exposure on mIPSC decay time. Averaged mIPSCs are shown from a neuron exposed to 50 mM ethanol for 4 hours (Ethanol) and a control neuron (Control) for which the ethanol was replaced by standard culture media. Current amplitudes from the two neurons were standardized and expressed as a percent of the peak response for each neuron to illustrate the decrease in decay time following a 4 hour ethanol exposure. Details of the decay-tau, amplitude and rate parameters averaged across several neurons are shown in Table 1.

Figure 4

Effects of PKC on GABA_A receptor α4 subunit expression. A. Cultured cortical neurons were exposed to PDBu (100nM) for 1hr and P2 fractions were isolated and analyzed by western blot analysis. PDBu increased α4 expression similar to ethanol. Data represent mean ± S.E.M. (Student's t-test, t = 7.954, * p < 0.05, n=4/group). B. Neurons were exposed to ethanol to 4 hrs in the presence or absence of the PKC inhibitor calphostin C (0.3μM). Calphostin C reversed ethanol-induced increases in α4 subunit expression. Calphostin C alone had no effect of α4 subunit expression. Data represent mean ± S.E.M. (anova, F = 9.882, p < 0.01, Newman-Keuls posthoc test * p < 0.05,), n=6/group. Representative western blot images are shown.

Figure 5

Effects of specific PKC isoforms on GABA_A receptor α4 subunit expression in the P2 fraction. A. Cultured cortical neurons were transfected with scrambled siRNA or siRNA specific for PKCγ. PKCγ siRNA inhibited ethanol-induced increases in α4 subunit expression. PKCγ siRNA alone had no effect on α4 subunit expression. Data represent mean ± S.E.M. *, p < 0.05 compared to vehicle, (anova, F = 4.265, p < 0.05, Newman-Keuls posthoc test), n = 6-7/group. B. Cultured neurons were exposed to PKCβ inhibitor PKCβ pseudosubstrate (pseudo) alone or in the presence of ethanol. Blocking PKCβ had no effect on ethanol-induced increases in α4 subunit expression. PKCβ pseudosubstrate alone had no effect on α4 subunit expression. Data represent mean ± S.E.M. *, p < 0.05 vs. Vehicle (anova, F = 4.015, Newman-Keuls posthoc test), n = 4-6/group. Representative western blot images are shown.