Abstract
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 GABAA receptors composed of α1β2/3γ2 subunits. However, the mechanisms of ethanol-mediated internalization are unknown. This study explored the effect of ethanol on surface expression of GABAA α1 subunit-containing receptors in cultured cerebral cortical neurons and the role of protein kinase C (PKC) β, γ, and ε 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 GABAA receptor α1 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 PKCγ with α1 subunits, whereas PKCβ/α1 association and PKCε/α1 colocalization were not altered by ethanol exposure. Selective PKCγ inhibition by transfection of selective PKCγ small interfering RNAs blocked ethanol-induced internalization of GABAA receptor α1 subunits, whereas PKCβ inhibition using pseudo-PKCβ had no effect. These findings suggest that ethanol exposure selectively alters PKCγ translocation to GABAA receptors and PKCγ regulates GABAA α1 receptor trafficking after ethanol exposure.
Footnotes
This work was supported by the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grants AA015409 (to S.K.), AA11605 (to A.L.M.)].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.063016.
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ABBREVIATIONS:
- CCV
- clathrin-coated vesicle
- PKC
- protein kinase C
- siRNA
- small interfering RNA
- Pen-Strep
- penicillin-streptomycin
- PBS
- phosphate-buffered saline
- ANOVA
- analysis of variance
- BSA
- bovine serum albumin
- PAGE
- polyacrylamide gel electrophoresis
- PDBu
- phorbol-12,13-dibutyrate.
- Received December 8, 2009.
- Accepted February 16, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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