The long-term objective of my work is to provide a better understanding of synaptic transmission by studying the operation of NMDA, AMPA and kainate receptors, which form ion channels gated by the neurotransmitter glutamate. Another major goal is to uncover properties of these receptors that may allow for clinical intervention to prevent excitotoxic cell death or to provide analgesia. The experiments in this proposal arise from two interesting discoveries that we made during the current period of support.
Specific Aim 1 follows up on our observation that kainate receptor block by cis-unsaturated fatty acids, such as docosahexanoic acid (DHA), depends on RNA editing at the Q/R site in the channel pore. Channels with subunits in the edited (R) form are strongly blocked, but inclusion of subunits with a (Q) at the Q/R site dramatically weakens inhibition. Site-directed mutagenesis in the pore loop segment will be used to test the structural basis for this regulation of recombinant channels.
Specific Aim 2 builds on our discovery that mutations near the extracellular end of the M3 helix regulate fatty acid block. Chimeric subunits and point mutations will be used to determine the structural requirements for fatty acid inhibition in domains outside of the pore loop segment tested in Aim 1.
Specific Aim 3 will analyze channel modulation resulting from release of endogenous lipid-derived mediators. Stimuli relevant to normal brain electrical activity, and to a variety of brain pathologies associated with fatty acid release, will be used to trigger lipid mobilization. The experiments will make use of subunits that resist fatty acid modulation as well as small molecule antagonists that we have discovered which block fatty acid potentiation of NMDA receptors. Collectively, these experiments will shed light on the operation and regulation of neuronal glutamate receptors. Subunits that make up these receptors are all homologous to each other and are thought to share the same membrane topology. Structural differences among the subunits underlie the unique contributions made by NMDA, AMPA and kainate receptors to synaptic communication. Our experiments focus on structural features that allow for differential regulation of receptors with specific subunit composition. A number of pathologic conditions, including brain trauma, epilepsy, and ischemia, elicit massive release of cis-unsaturated fatty acids. These compounds directly regulate many different membrane proteins including a number of ion channel subtypes. This project analyzes the molecular basis of glutamate receptor modulation by DHA, which is present at high levels in the nervous system and is known to be essential for normal brain function.
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