Expression of the GluR2 subunit controls the calcium permeability and rectification properties of the AMPA subtype of glutamate receptor channels. A growing number of findings indicate that the mechanisms responsible for moderate changes in GluR2 expression, both developmentally and after traumatic insult in the adult, are important neuron-specific regulatory controls governing synaptic phenotype. The immediate objective is to identify and characterize the key mechanisms by which GluR2 expression is regulated in neurons. The applicants have cloned the 5' flanking sequence of the rat GluR2 gene and propose a series of experiments to identify key regulatory elements that control neuron-specific expression. These experiments will involve functional analysis of promoter-reporter constructs transfected into neurons, electrophoretic mobility shift assays with known transcription factors, and in vitro and in vivo footprint analyses. Possible translational controls mediated by sequence elements in the 5' untranslated leader of the GluR2 mRNA will also be explored. The results obtained should permit exploration of the physiological consequences of selective down-regulation of GluR2 expression. To this end transcription factors targeting key regulatory elements in the GluR2 gene will be delivered to cultured neurons by adenovirus, and enhancer elements will be deleted from the endogenous promoter by in vivo gene targeting. The effects of selective down-regulation of GluR2 will be judged by a combination of electrophysiology, excitotoxicity assays, and fluorescence microscopy measurements of calcium handling. The results should improve our understanding of the physiological consequences of moderate variations in GluR2 expression, and may suggest new therapeutic strategies for several neurologic disorders based on adjusting the relative abundance of the GluR2 subunit.
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