Department of Psychiatry, Bowles Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Mol Pharmacol. 2010 May;77(5):793-803. doi: 10.1124/mol.109.063016. Epub 2010 Feb 16.
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.
长期乙醇暴露会导致中枢神经系统过度兴奋，涉及 GABA 能抑制的丧失。我们之前的研究表明，长期乙醇暴露会增强由α1β2/3γ2 亚基组成的突触 GABA(A)受体的内化。然而，乙醇介导的内化机制尚不清楚。本研究探讨了乙醇对培养大脑皮质神经元中 GABA(A)α1 亚基包含受体表面表达的影响，以及蛋白激酶 C (PKC)β、γ 和 ε 同工型在其运输中的作用。培养的神经元是从小鼠出生后第 1 天开始制备的，并维持 18 天。细胞暴露于乙醇中，通过生物素化和 P2 级分分离表面受体，而通过全细胞膜片钳记录 GABA 和唑吡坦诱导的反应进行功能分析。乙醇暴露 4 小时会降低 GABA(A)受体α1 亚基的生物素化表面表达，并减少唑吡坦（100 nM）对 GABA 诱导电流的增强作用。PKC 激活剂佛波醇-12,13-二丁酸酯模拟了乙醇的作用，而选择性 PKC 抑制剂钙调蛋白 C 可防止乙醇诱导这些受体的内化。乙醇暴露 4 小时还会增加 PKCγ与α1 亚基的共定位和共免疫沉淀，而乙醇暴露并未改变 PKCβ/α1 结合和 PKCε/α1 共定位。选择性 PKCγ 抑制通过转染选择性 PKCγ 小干扰 RNA 阻断了 GABA(A)受体α1 亚基的乙醇诱导内化，而使用假 PKCβ 抑制 PKCβ 则没有作用。这些发现表明，乙醇暴露选择性地改变了 PKCγ向 GABA(A)受体的易位，并且 PKCγ 在乙醇暴露后调节 GABA(A)α1 受体的运输。
