NMDA receptors (NMDARs) are glutamate receptor ion channels that mediate excitatory neurotransmission. The majority of NMDARs are composed of two GluN1 and two GluN2 subunits. One GluN1 subunit has been cloned, but there are four GluN2 subunits (GluN2A-D) that endow NMDARs with distinct functional properties and different developmental and regional expression. Mutations in the gene encoding the GluN2A subunit have been associated with childhood epilepsy/aphasia syndromes, and a subset of these mutations cause gain-of- function in GluN2A-containing receptors that lead to severe neurologic complications. The high frequency of GluN2A mutations linked to neurologic conditions provides a compelling rationale for the development of GluN2A-selective therapeutic agents. GluN2A-selective modulators can also be useful and much needed pharmacological tools for neurophysiological studies. We have identified novel GluN2A-selective negative allosteric modulators (NAMs) that inhibit NMDARs by reducing glycine binding to the GluN1 subunit. These NAMs can transform our ability to evaluate the contribution of GluN2A to normal brain function and disease. In addition, we have determined crystal structures of the heterodimer formed by GluN1 and GluN2A agonist binding domains (ABDs) with NAMs bound at the subunit interface. These crystal structures represent the discovery of a novel, unexplored modulatory site and provide new opportunities for the development of subunit- selective ligands. We will uncover structural and mechanistic features of this modulatory site and provide important understanding required for the use of GluN2A-selective NAMs as pharmacological tools.
Aim 1) What are the structural bases for selectivity, potency, and efficacy of GluN2A-selective NAMs? We will use crystallography, mutagenesis, and electrophysiological recordings of NMDA receptor function to define the structural determinants for NAM inhibition.
Aim 2) What is the mechanistic basis for allosteric interaction between NAM and glycine binding sites? Crystallographic, pharmacological, and mutational analyses will dissect the conformational changes and mechanism that causes the allosteric interaction between NAM and glycine binding sites.
Aim 3) Can GluN2A-selective NAMs inhibit triheteromeric and neuronal NMDA receptors? We will define NAM inhibition of NMDARs in conditions relevant to synaptic transmission as well as NAM inhibition of NMDARs that contain gain-of-function GluN2A mutations identified in epilepsy patients. Neuronal NMDARs are heterogeneous populations of diheteromeric receptors comprised of two GluN1 and two identical GluN2 subunits (e.g. GluN1/GluN2A) as well as triheteromeric receptors containing two GluN1 and two different GluN2 subunits (e.g. GluN1/GluN2A/GluN2B). Evaluation of GluN2A-selective modulators on both diheteromeric and triheteromeric receptors is required for their use as pharmacological tools. In addition, we will inhibit NMDAR-mediated synaptic currents in primary cultures of autaptic neurons that express different ratios of GluN2A and GluN2B as well as autaptic neurons that express GluN2A with gain-of-function mutations.

Public Health Relevance

NMDA receptors mediate excitatory neurotransmission in the central nervous system and play important roles in neuronal development and synaptic plasticity. They are also implicated in many neurological and psychiatric disorders, and subunit-selective modulation of NMDA receptors by targeting a single GluN2 subunit in specific brain regions or neuronal cell types could be therapeutically beneficial in a number of diseases. We will determine the structural and mechanistic bases for selective inhibition of GluN2A-containing NMDA receptors in conditions relevant to synaptic transmission and childhood epilepsy disorders caused by gain-of-function mutations in the gene encoding the GluN2A subunit.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS097536-02
Application #
9317543
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Silberberg, Shai D
Project Start
2016-08-01
Project End
2021-05-31
Budget Start
2017-06-01
Budget End
2018-05-31
Support Year
2
Fiscal Year
2017
Total Cost
$317,188
Indirect Cost
$98,438
Name
University of Montana
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
010379790
City
Missoula
State
MT
Country
United States
Zip Code
59812
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Yi, Feng; Zachariassen, Linda G; Dorsett, Katherine N et al. (2018) Properties of Triheteromeric N-Methyl-d-Aspartate Receptors Containing Two Distinct GluN1 Isoforms. Mol Pharmacol 93:453-467
Bhattacharya, Subhrajit; Khatri, Alpa; Swanger, Sharon A et al. (2018) Triheteromeric GluN1/GluN2A/GluN2C NMDARs with Unique Single-Channel Properties Are the Dominant Receptor Population in Cerebellar Granule Cells. Neuron 99:315-328.e5
Gynther, Mikko; Proietti Silvestri, Ilaria; Hansen, Jacob C et al. (2017) Augmentation of Anticancer Drug Efficacy in Murine Hepatocellular Carcinoma Cells by a Peripherally Acting Competitive N-Methyl-d-aspartate (NMDA) Receptor Antagonist. J Med Chem 60:9885-9904
Lind, Genevieve E; Mou, Tung-Chung; Tamborini, Lucia et al. (2017) Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits. Proc Natl Acad Sci U S A 114:E6942-E6951
Jessen, Maja; Frederiksen, Kristen; Yi, Feng et al. (2017) Identification of AICP as a GluN2C-Selective N-Methyl-d-Aspartate Receptor Superagonist at the GluN1 Glycine Site. Mol Pharmacol 92:151-161
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Yi, Feng; Traynelis, Stephen F; Hansen, Kasper B (2017) Selective Cell-Surface Expression of Triheteromeric NMDA Receptors. Methods Mol Biol 1677:145-162
Sun, Weinan; Hansen, Kasper B; Jahr, Craig E (2017) Allosteric Interactions between NMDA Receptor Subunits Shape the Developmental Shift in Channel Properties. Neuron 94:58-64.e3
Tamborini, Lucia; Chen, Ying; Foss, Catherine A et al. (2016) Development of Radiolabeled Ligands Targeting the Glutamate Binding Site of the N-Methyl-d-aspartate Receptor as Potential Imaging Agents for Brain. J Med Chem 59:11110-11119

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