NMDA RECEPTORS, PROTEIN KINASE C, AND EXCITOTOXICITY

兴奋性毒性是许多神经系统疾病的主要病理生理机制，它是由一种特殊类型的谷氨酸受体--N-甲基-D-天冬氨酸(NMDA)受体介导的。了解NMDA受体的激活对于开发阻断兴奋性毒性的治疗策略至关重要。NMDA受体在大脑中以许多不同的亚型存在，其组成因存在的亚基类型而不同。内源性酶蛋白激酶C在大脑中异质性地调节NMDA受体的功能，可能反映了NMDA受体组成的不同。在异源表达系统中，NMDA受体的两种亚基(NR1和NR2)对PKC的调节作用不同。在目前的研究中，我们将确定这种调制发生的机制，以及在异源系统中的发现是否解释了在大脑中发现的PKC作用的多样性。在第一个目标中，我们将使用生化和分子生物学的方法来确定NR2A亚基上被PKC磷酸化的一个或多个确切位置。这将从体外的磷酸化实验开始。定点突变将可以确定这些位点是否与PKC对NMDA受体的NR2A型依赖的增强有因果联系。在第二个目标中，我们将确定PKC介导NR1依赖的NMDA受体减弱的细胞机制。具体的实验将检验改变钙调蛋白、肌动蛋白和中间细丝的试剂的效果，所有这些都以亚型特有的方式与NR1相互作用。在我们的第三个目标中，我们将确定PKC对天然NMDA受体的细胞特异性影响是否反映了NMDA受体类型的细胞特异性差异。这些实验将使用测量PKC对单个细胞的NMDA受体的影响，并使用RNA扩增技术来确定在这些特定的细胞中产生了哪些NMDA受体mRNAs。制造的NMDA受体mRNAs的类型将与细胞反应定量相关。在我们的最终目标中，我们将使用兴奋性毒性模型来确定PKC在增强或抑制NMDA受体介导的兴奋性毒性中所起的作用。这一系统的方法应该提供关于PKC控制NMDA受体的完整机制及其在生理和病理生理过程中的意义的关键信息。

Excitotoxicity, a major pathophysiological mechanism of many neurological disorders, is mediated by a specific type of glutamate receptor, the n-methyl-D-aspartate (NMDA) receptor. Understanding NMDA receptor activation is crucial for developing therapeutic strategies which block excitotoxicity. The NMDA receptor is present in brain as many different subtypes, which differ in composition by the type of subunits which are present. The endogenous enzyme protein kinase C modulates NMDA receptor function heterogeneously in the brain, perhaps reflecting varying composition of NMDA receptors. In heterologous expression systems, the two types of NMDA receptor subunits (NR 1 and NR2) subunit contribute differently to modulation by PKC. In the present study, we will determine the mechanisms by which such modulation occurs and whether findings in heterologous systems explain the diversity of effects of PKC found in the brain. In the first aim, we will use biochemical and molecular biological approaches to determine the exact site or sites on the NR2A subunit which are phosphorylated by PKC. This will begin with phosphorylation experiments in vitro. Site directed mutations will allow determination of whether these sites are causally linked with NR 2A-dependent potentiation of NMDA receptors by PKC. In the second aim we will ascertain the cellular mechanism which mediates NR1 dependent attenuation of NMDA receptors by PKC. Specific experiments will examine the effects of agents which alter calmodulin, actin, and intermediate filaments, all of which interact with NR 1 in subtype specific manners. In our third aim we will determine whether the cell specific effects of PKC on native NMDA receptors reflect cell specific differences in the type of NMDA receptors which are made. These experiments will use measurements of the effect of PKC on NMDA receptors of single cells, and use RNA amplification techniques to determine which NMDA receptors mRNAs are made in those specific cells. The type of NMDA receptor mRNAs made will be correlated quantitatively with the cellular response. In our final aim we will use a model of excitotoxicity to determine the role which PKC plays in potentiation or inhibition of NMDA receptor mediated excitotoxicity. This systematic approach should provide critical information on the complete mechanisms of control of NMDA receptors by PKC and their significance in physiologic and pathophysiologic processes.