NMDA RECEPTORS, PROTEIN KINASE C, AND EXCITOTOXICITY

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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.