Thousands of lives are devastated each year by spinal cord injury. A significant portion of the motor deficits and pain experienced by these patients could be prevented if therapies were devised to block posttrauma degradation of the surviving tissue. Trauma to the spinal cord initiates a cascade of biochemical events that exacerbate the injury. The extended damage leads to an additional decline in motor function and to sensory disturbances such as chronic pain. As part of these processes the opioid peptide dynorphin increases in concentration and is released in the spinal cord during the post-trauma period. Dynorphin was previously demonstrated to enhance N-methyl-D-aspartate (NMDA) receptor function to produce neuronal damage in the spinal cord. Thus, dynorphin is likely to be a significant participant in the post-insult degenerative events. This study will test the hypothesis that dynorphin binds to NR1 splice variants that do not possess the exon-5 coded region of the protein (NRla) to expose previously inactive or low activity NMDA receptors. A second hypothesis that will be tested is that dynorphin induces migration of internal stores of NMDA receptors to the plasma membrane. To test these hypotheses three aims are proposed. In the first aim Hek-293 cells will be transiently transfected with the 32 possible pairs of NR1 splice variants and NR2 subunits. The sensitivity of the various subunit combinations to dynorphin and NMDA induced excitotoxicity will be evaluated by measuring the release of lactate dehydrogenase into the culture media using a high throughput 96 well assay. In the second aim the combinations of NR1 and NR2 subunits that are most sensitive to dynorphin (determined from aim 1) will be examined using whole cell patch clamp and cell surface labeling techniques to determine how dynorphin influences the receptor's function. In the final aim the pairs of NR1 splice variants and NR2 subunits expressed in the spinal cord will be determined using co-immunoprecipitation, 2D-electrophoresis, immunohistochemistry and RT-PCR. These experiments will provide the subunit identity of the dynorphin enhanced NMDA receptors in the spinal cord and the mechanism for how dynorphin enhances NMDA receptor function. It is hoped that this information will provide a valuable pharmacological target for interrupting dynorphin's participation in post spinal trauma neurodegeneration.
