Xu Minfu, Woodward John J
Department of Neuroscience and Center for Drug and Alcohol Programs, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Neurochem. 2006 Mar;96(6):1760-7. doi: 10.1111/j.1471-4159.2006.03703.x.
N-methyl-D-aspartate receptors (NMDA) are glutamate-activated ligand-gated ion channels that participate in diverse forms of synaptic plasticity as well as glutamate-dependent excitotoxicity. Inhibition of the NMDA receptor function may underlie some of the behavioral actions associated with acute exposure to ethanol. The sensitivity of NMDA receptors to ethanol is influenced by the subunit composition of the receptor and, by association, with certain cytoskeletal proteins. Previous studies have also suggested that phosphorylation may regulate the sensitivity of NMDA receptors to ethanol. In this study, the ethanol inhibition of recombinant NMDA receptor currents was determined under conditions designed to enhance or inhibit the activity of protein kinase A (PKA). Human embryonic kidney 293 (HEK293) cells were transfected with cDNAs encoding NMDA subunits and channel activity was monitored with whole-cell patch-clamp electrophysiology. Under control recording conditions, ethanol (100 mM) inhibited NR1/2A and NR1/2B receptor currents by approximately 25-30%. The degree of ethanol inhibition was not affected or was slightly enhanced under conditions designed to enhance PKA activity. This included treatment of cells with cAMP analogs, inclusion of phosphatase inhibitors or purified PKA in the pipette filling solution, co-expression of catalytically active PKA, expression of the NR1 PKA-site phosphorylation site mimic (S897D) or by co-expression of the PKA scaffolding protein yotiao or the dopamine D(1) receptor. Ethanol inhibition of NR1/2A and NR1/2B receptors was not altered when PKA effects were suppressed, either by co-expression of a PKI inhibitory peptide or the phosphorylation-deficient NR1 mutants (S897A, S896A, S896A/S897A). In addition, ethanol inhibition of NMDA-induced currents in cultured cortical or hippocampal neurons was not affected by modulators of PKA. These results suggest that PKA does not appear to play a major role in determining the acute ethanol sensitivity of NMDA receptors.
N-甲基-D-天冬氨酸受体（NMDA）是谷氨酸激活的配体门控离子通道，参与多种形式的突触可塑性以及谷氨酸依赖性兴奋毒性。抑制NMDA受体功能可能是急性接触乙醇相关的一些行为作用的基础。NMDA受体对乙醇的敏感性受受体亚基组成以及与某些细胞骨架蛋白结合的影响。先前的研究还表明，磷酸化可能调节NMDA受体对乙醇的敏感性。在本研究中，在旨在增强或抑制蛋白激酶A（PKA）活性的条件下，测定了乙醇对重组NMDA受体电流的抑制作用。用编码NMDA亚基的cDNA转染人胚肾293（HEK293）细胞，并用全细胞膜片钳电生理学监测通道活性。在对照记录条件下，乙醇（100 mM）抑制NR1/2A和NR1/2B受体电流约25%-30%。在旨在增强PKA活性的条件下，乙醇抑制程度未受影响或略有增强。这包括用cAMP类似物处理细胞、在移液管填充溶液中加入磷酸酶抑制剂或纯化的PKA、共表达具有催化活性的PKA、表达NR1 PKA位点磷酸化位点模拟物（S897D）或共表达PKA支架蛋白yotiao或多巴胺D(1)受体。当通过共表达PKI抑制肽或磷酸化缺陷型NR1突变体（S897A、S896A、S896A/S897A）抑制PKA效应时，乙醇对NR1/2A和NR1/2B受体的抑制作用未改变。此外，PKA的调节剂不影响乙醇对培养的皮质或海马神经元中NMDA诱导电流的抑制作用。这些结果表明，PKA似乎在决定NMDA受体的急性乙醇敏感性方面不起主要作用。
