EXPRESSION AND REGULATION OF KAINATE RECEPTORS

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Glutamate, the major excitatory neurotransmitter in the mammalian CNS, gates three major types of ionotropic receptors, named NMDA, AMPA and kainate, based on their pharmacology and molecular properties of the receptor subunits. This proposal will focus on the kainate receptor of which five subunits have been cloned so far: GluR5, GluR6, GluR7, KA1 and KA2. Although these subunits are expressed throughout the brain, fast- desensitizing kainate receptors have only been found in the dorsal root ganglion (Huettner, 1990). In this study, we will investigate the subunit composition of native kainate receptors, how the biophysical properties can be regulated by different subunit forms, and the role of phosphorylation in the modulation of channel properties. Kainate receptors are believed to play a key role in mediating fast synaptic transmission and may regulate Ca2+ entry during development. Therefore, a better understanding of their structure, function and modulation will provide insight into their role in synaptic plasticity and cell death. The developmental expression of kainate receptor subunit RNA and protein will be studied in cerebellar granule cells to identify which isoforms are present in this single neuronal type. Patchclamp experiments will determine the functional properties of these channels recombinantly expressed in HEK293 cells. For this project, we will primarily use cloned kainate receptor cDNAs stably integrated into the genome or transiently transfected into HEK293 cells. The stable expression system provides additional and necessary information to cerebellar granule cells because a single population of identical channels can be studied in the absence of other glutamate-receptor gene products. Biochemical studies on the effect of phosphorylation on kainate receptor expression will be performed on the stable cell lines, as well as cerebellar granule cells to identify functional differences. The advantage of stable cells and cerebellar granule cells is that large quantities of kainate receptors can be obtained for biochemical analysis of the regulation of the level of surface expression and phosphorylation. Working together, the two approaches can provide powerful insights into the molecular mechanisms that underlie kainate receptor function in the CNS.