Carlson Stephen L, Bohnsack John Peyton, Morrow A Leslie
Departments of Psychiatry and Pharmacology, Bowles Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, North Carolina.
Departments of Psychiatry and Pharmacology, Bowles Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, North Carolina
J Pharmacol Exp Ther. 2016 Apr;357(1):10-6. doi: 10.1124/jpet.115.230417. Epub 2016 Feb 8.
Ethanol alters GABAA receptor trafficking and function through activation of protein kinases, and these changes may underlie ethanol dependence and withdrawal. In this study, we used subsynaptic fraction techniques and patch-clamp electrophysiology to investigate the biochemical and functional effects of protein kinase A (PKA) and protein kinase C (PKC) activation by ethanol on synaptic GABAA α4 receptors, a key target of ethanol-induced changes. Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or kinase modulators for 4 hours, a paradigm that recapitulates GABAergic changes found after chronic ethanol exposure in vivo. PKA activation by forskolin or rolipram during ethanol exposure prevented increases in P2 fraction α4 subunit abundance, whereas inhibiting PKA had no effect. Similarly, in the synaptic fraction, activation of PKA by rolipram in the presence of ethanol prevented the increase in synaptic α4 subunit abundance, whereas inhibiting PKA in the presence of ethanol was ineffective. Conversely, PKC inhibition in the presence of ethanol prevented the ethanol-induced increases in synaptic α4 subunit abundance. Finally, we found that either activating PKA or inhibiting PKC in the presence of ethanol prevented the ethanol-induced decrease in GABA miniature inhibitory postsynaptic current decay τ1, whereas inhibiting PKA had no effect. We conclude that PKA and PKC have opposing effects in the regulation of synaptic α4 receptors, with PKA activation negatively modulating, and PKC activation positively modulating, synaptic α4 subunit abundance and function. These results suggest potential targets for restoring normal GABAergic functioning in the treatment of alcohol use disorders.
乙醇通过激活蛋白激酶改变GABAA受体的转运和功能，而这些变化可能是乙醇依赖和戒断的基础。在本研究中，我们使用突触下组分技术和膜片钳电生理学方法，研究乙醇激活蛋白激酶A（PKA）和蛋白激酶C（PKC）对突触GABAAα4受体的生化和功能影响，该受体是乙醇诱导变化的关键靶点。大鼠大脑皮质神经元在体外培养18天，然后暴露于乙醇和/或激酶调节剂4小时，该模式概括了体内慢性乙醇暴露后发现的GABA能变化。在乙醇暴露期间，用福斯可林或咯利普兰激活PKA可防止P2组分α4亚基丰度增加，而抑制PKA则无效。同样，在突触组分中，在乙醇存在下用咯利普兰激活PKA可防止突触α4亚基丰度增加，而在乙醇存在下抑制PKA则无效。相反，在乙醇存在下抑制PKC可防止乙醇诱导的突触α4亚基丰度增加。最后，我们发现，在乙醇存在下激活PKA或抑制PKC均可防止乙醇诱导的GABA微小抑制性突触后电流衰减τ1降低，而抑制PKA则无效。我们得出结论，PKA和PKC在突触α4受体的调节中具有相反的作用，PKA激活对突触α4亚基丰度和功能起负调节作用，而PKC激活起正调节作用。这些结果提示了在酒精使用障碍治疗中恢复正常GABA能功能的潜在靶点。
