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.
Kumar
Sandeep
S
Department of Psychiatry, Bowles Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Suryanarayanan
Asha
A
Boyd
Kevin N
KN
Comerford
Chris E
CE
Lai
Marvin A
MA
Ren
Qinglu
Q
Morrow
A Leslie
AL
D000818:Animals; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000187:drug effects / Q000502:physiology; D017931:DNA Primers; D000431:Ethanol / Q000494:pharmacology*; D018755:GABA Agonists / Q000494:pharmacology; D008578:Meninges / Q000187:drug effects / Q000502:physiology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000502:physiology*; D000077334:Zolpidem
D017931:DNA Primers; D018755:GABA Agonists; C546242:Gabra1 protein, rat; D011725:Pyridines; D034741:RNA, Small Interfering; D011963:Receptors, GABA-A; D000431:Ethanol; D000077334:Zolpidem; C087857:protein kinase C gamma; D011493:Protein Kinase C