PKCγ is required for ethanol-induced increases in GABAA receptor α4 subunit expression in cultured cerebral cortical neurons

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