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.
Department of Psychiatry, Bowles Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Sandeep
S
Kumar
Asha
A
Suryanarayanan
Kevin N
KN
Boyd
Chris E
CE
Comerford
Marvin A
MA
Lai
Qinglu
Q
Ren
A Leslie
AL
Morrow
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