Ethanol reduces GABAA alpha1 subunit receptor surface expression by a protein kinase Cgamma-dependent mechanism in cultured cerebral cortical neurons

Prolonged ethanol exposure causes central nervous system hyperexcitability that involves a loss of GABAergic inhibition. We previously demonstrated that long-term ethanol exposure enhances the internalization of synaptic GABA(A) receptors composed of alpha1beta2/3gamma2 subunits. However, the mechanisms of ethanol-mediated internalization are unknown. This study explored the effect of ethanol on surface expression of GABA(A) alpha1 subunit-containing receptors in cultured cerebral cortical neurons and the role of protein kinase C (PKC) beta, gamma, and epsilon isoforms in their trafficking. Cultured neurons were prepared from rat pups on postnatal day 1 and maintained for 18 days. Cells were exposed to ethanol, and surface receptors were isolated by biotinylation and P2 fractionation, whereas functional analysis was conducted by whole-cell patch-clamp recording of GABA- and zolpidem-evoked responses. Ethanol exposure for 4 h decreased biotinylated surface expression of GABA(A) receptor alpha1 subunits and reduced zolpidem (100 nM) enhancement of GABA-evoked currents. The PKC activator phorbol-12,13-dibutyrate mimicked the effect of ethanol, and the selective PKC inhibitor calphostin C prevented ethanol-induced internalization of these receptors. Ethanol exposure for 4 h also increased the colocalization and coimmunoprecipitation of PKCgamma with alpha1 subunits, whereas PKCbeta/alpha1 association and PKCepsilon/alpha1 colocalization were not altered by ethanol exposure. Selective PKCgamma inhibition by transfection of selective PKCgamma small interfering RNAs blocked ethanol-induced internalization of GABA(A) receptor alpha1 subunits, whereas PKCbeta inhibition using pseudo-PKCbeta had no effect. These findings suggest that ethanol exposure selectively alters PKCgamma translocation to GABA(A) receptors and PKCgamma regulates GABA(A) alpha1 receptor trafficking after ethanol exposure.