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 several interesting discoveries that we made during the current period of support.
Specific Aim 1 follows up on our observation that interactions between the pore loop and adjacent transmembrane helices govern kainate receptor susceptibility to inhibition by cis-unsaturated fatty acids, such as docosahexaenoic acid (DHA). Experiments in this aim will use mutant cycle analysis to determine which residues in the channel interact with each other to control permeation, gating and modulation.
Specific Aim 2 builds on our discovery that exposure to DHA appears to change the conformation of kainate receptor transmembrane helices in the open state. Reactivity of substituted cysteines, metal ion binding, and disulfide bond formation will be used to determine structural changes introduced by fatty acids.
Specific Aim 3 follows up on our discovery of small molecule antagonists that prevent DHA from potentiating NMDA receptors but have little or no effect on DHA inhibition of kainate receptors. Chimeric subunits that combine domains from NMDA and kainate receptors will be used to investigate whether distinct structural interactions underlie the potentiation and inhibition of channel activity and t analyze the structural requirements for generation of functional channels. Collectively, these experiments will provide new information about the structural basis for ionotropic glutamate receptor operation and new information about how ion channels are affected by interactions with components of the lipid bilayer. 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.

Public Health Relevance

Traumatic brain injury, ischemia and epilepsy, as well as normal brain electrical activity, stimulate the release of free cis-unsaturated fatty acids from neuronal membrane phospholipids. In addition, abnormal lipid metabolism and selective reductions in brain cis-unsaturated fatty acid levels are associated with neurodegenerative diseases such as Alzheimer's disease. This project analyzes how these fatty acids control the operation of ion channels gated by L-glutamate, the main central nervous system excitatory neurotransmitter.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS030888-18A1
Application #
8370043
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Whittemore, Vicky R
Project Start
1992-07-01
Project End
2017-04-30
Budget Start
2012-05-15
Budget End
2013-04-30
Support Year
18
Fiscal Year
2012
Total Cost
$332,500
Indirect Cost
$113,750
Name
Washington University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Wilding, Timothy J; Lopez, Melany N; Huettner, James E (2016) Chimeric Glutamate Receptor Subunits Reveal the Transmembrane Domain Is Sufficient for NMDA Receptor Pore Properties but Some Positive Allosteric Modulators Require Additional Domains. J Neurosci 36:8815-25
Iyer, Nisha R; Huettner, James E; Butts, Jessica C et al. (2016) Generation of highly enriched V2a interneurons from mouse embryonic stem cells. Exp Neurol 277:305-16
Xu, Hao; Iyer, Nisha; Huettner, James E et al. (2015) A puromycin selectable cell line for the enrichment of mouse embryonic stem cell-derived V3 interneurons. Stem Cell Res Ther 6:220
Huettner, James E (2015) Glutamate receptor pores. J Physiol 593:49-59
McCreedy, Dylan A; Brown, Chelsea R; Butts, Jessica C et al. (2014) A new method for generating high purity motoneurons from mouse embryonic stem cells. Biotechnol Bioeng 111:2041-55
Huettner, James E (2014) Vertebrate galectins: endogenous regulators of ionotropic glutamate receptors? J Physiol 592:2035-6
Wilding, Timothy J; Lopez, Melany N; Huettner, James E (2014) Radial symmetry in a chimeric glutamate receptor pore. Nat Commun 5:3349
Chandrasekar, Indra; Huettner, James E; Turney, Stephen G et al. (2013) Myosin II regulates activity dependent compensatory endocytosis at central synapses. J Neurosci 33:16131-45
Lopez, Melany N; Wilding, Timothy J; Huettner, James E (2013) Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles. J Gen Physiol 142:225-39
Solomon, Isaac H; Khatri, Natasha; Biasini, Emiliano et al. (2011) An N-terminal polybasic domain and cell surface localization are required for mutant prion protein toxicity. J Biol Chem 286:14724-36

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