Rapid communication between neurons is accomplished by the action of neurotransmitters on postsynaptic receptors. At excitatory synapses, these receptors belong to the family of ionotropic glutamate receptors. By regulating the activity and membrane density of glutamate-gated ion channels, neurons alter the strength of their synaptic inputs in response to developmental cues and sensory stimuli. Both inadequate and excessive activity of glutamate receptors has been linked to psychiatric disease and neurological deficit. Determining molecular features of glutamate receptor regulation is crucial for our understanding of normal excitatory synapse function, and should facilitate the design of rational strategies for the treatment of neurologic and psychiatric disease. The objective of this proposal is to understand how neurons assemble and regulate the postsynaptic molecular machinery present at excitatory synapses. In particular, the proposed research will determine molecular mechanisms which regulate the synaptic targeting and clustering of the N-methyl-D-aspartate (NMDA) class of ionotropic glutamate receptors. We have recently identified cytoplasmic domains of the NMDA receptor subunit NR1 important for receptor targeting and receptor inactivation. These regions of NR1 are thought to mediate their effects by interacting with cytoskeletal proteins and signaling molecules. To further elucidate the function of NMDA receptor cytoplasmic domains, we will first identify novel molecules which interact with NR1 subunits using both yeast two-hybrid screening and biochemical approaches. Second, the effects of these interactions on NMDA receptor channel activity and synaptic localization will be directly tested by electrophysiological and immunocytochemical methods. Third, NMDA receptor targeting motifs will be identified using fluorescent localization of tagged wild-type and mutant NR1 and NR2 subunit cytoplasmic domains expressed in cultured neurons. Finally, the effect of synaptic activity on the interaction of cellular proteins with native NMDA receptors will be assessed using covalent crosslinking techniques. Together, the proposed experiments will elucidate basic molecular mechanisms underlying synaptic transmission and synaptic plasticity.
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