Protein Kinase Cϵ Regulates γ-Aminobutyrate Type A Receptor Sensitivity to Ethanol and Benzodiazepines through Phosphorylation of γ2 Subunits

Chao Zhang,Wen-Hai Chou,Philip M. Newton,Maengseok Song,Melisa J. Wallace,Kevan M. Shokat,Robert O. Messing,Zhan-Heng Qi,Thomas McMahon,Dan Wang

doi:10.1074/jbc.m707233200

Abstract

Ethanol enhances gamma-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA(A) receptors, especially at low concentrations achieved during social drinking. We postulated that the epsilon isoform of protein kinase C (PKCepsilon) regulates the ethanol sensitivity of GABA(A) receptors, as mice lacking PKCepsilon show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCepsilon mutant to selectively inhibit the catalytic activity of PKCepsilon. We used this mutant and PKCepsilon(-/-) mice to determine that PKCepsilon phosphorylates gamma2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at alpha1beta2gamma2 receptors, which is the most abundant GABA(A) receptor subtype in the brain. Our findings indicate that PKCepsilon phosphorylation of gamma2 regulates the response of GABA(A) receptors to specific allosteric modulators, and, in particular, PKCepsilon inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.

Highlights

␥-Aminobutyrate type A (GABAA)3 receptors mediate the majority of rapid inhibitory neurotransmission in the brain and are an important target for ethanol, the most widely abused drug [1]
Because behavioral responses to benzodiazepines are increased in these mice [13], we tested their response to zolpidem, an imidazopyridine that selectively acts at the benzodiazepine site on GABAA receptors that contain ␣1 and ␥2 subunits [22]
We found that protein kinase C⑀ (PKC⑀)Ϫ/Ϫ mice showed greater rotarod ataxia in response to 1 mg/kg zolpidem than wild-type littermates (Fig. 1A)

Summary

Introduction

␥-Aminobutyrate type A (GABAA)3 receptors mediate the majority of rapid inhibitory neurotransmission in the brain and are an important target for ethanol, the most widely abused drug [1]. To test the effect of inhibiting PKC⑀ on GABAA receptor function, cells were incubated with external solution containing 1 ␮M 1-naphthyl-PP1 (1Na) or vehicle (0.1% Me2SO) for 30 min prior to stimulation with GABA. Increased Response to Zolpidem in PKC⑀Ϫ/Ϫ Mice—To determine whether PKC⑀ regulates GABAA receptors that contain ␣1 and ␥2 subunits, we first examined behavioral responses in mice that lack PKC⑀.

Results

Conclusion