title: 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.
authors.affiliation: Bowles Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
authors.forename: David F
authors.initials: DF
authors.lastname: Werner
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.forename: Hugh E
authors.initials: HE
authors.lastname: Criswell
authors.forename: Asha
authors.initials: A
authors.lastname: Suryanarayanan
authors.forename: J Alex
authors.initials: JA
authors.lastname: Fetzer
authors.forename: Chris E
authors.initials: CE
authors.lastname: Comerford
authors.forename: A Leslie
authors.initials: AL
authors.lastname: Morrow
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000096:biosynthesis*
chemical_list: C546262:Gabra4 protein, rat; D011963:Receptors, GABA-A; D000431:Ethanol; C087857:protein kinase C gamma; D011493:Protein Kinase C