The ionotropic glutamate receptors family includes the N-methyl D-aspartate (NMDA), alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, and delta receptors, mediate the excitatory synaptic transmission in the central nervous system. The NMDA receptor is a heteromeric receptor formed by two glycine binding GluN1 and two glutamate binding GluN2 subunits. The GluN2 subunit, of which there are four (GluN2A, B, C, and D) mediate the ion channel properties of the NMDA receptors. The GluN1/GluN2C NMDA receptor is highly expressed in the cerebellum, specifically on the cerebellar granule cell at the mossy fiber-cerebellar granule cell synapse. Less than 2% of the papers on NMDA receptors are on the GluN1/GluN2C receptor, probably because there has been a lack of pharmacological tools that can specifically identify the GluN1/GluN2C receptors in vivo. This application presents the first known compound class (ketopyrrolines) that selectively potentiates the GluN2C containing receptors. This application aims to identify the structural determinates and mechanism of action of ketopyrrolines on the GluN2C receptors, as well as the role of GluN2C in the induction of LTP in the cerebellum. These experiments will determine ketopyrrolines usefulness as a pharmacological tool to understand the role of GluN2C in the CNS.
Aim 1 : What are the possible targets and structural determinants of ketopyrrolines? Ketopyrrolines will be tested for GluN2C selectivity within the lab and at the NIMH funded Psychoactive Drug Screening Program at UNC. Gain of function and loss of function chimeras will be constructed between the GluN2A and Glun2C subunits expressed in Xenopus laevis oocytes. This technique was successfully used in the lab to identify the structural determinates for a GluN2C/D potentiator (CIQ) and inhibitor (QNZ46).
Aim 2 : What is the mechanism of action for ketopyrrolines? Whole-cell and single channel recording will be performed by expressing the GluN1/GluN2C receptor in a mammalian cell line. Ketopyrrolines will be applied by a fast perfusion system to measure its association, disassociation, open probability, and channel open and shut times.
Aim 3. How do the GluN2A and GluN2C subunits affect the threshold for plasticity in the cerebellum? The threshold for plasticity will be determined in the presence and absence of ketopyrrolines for the wild type, GluN2A and GluN2C knockout mice by varying the input stimulus. Patch Clamp recording will be carried out in cerebellar slices at an appropriate age for GluN2C expression. The successful completion of this application will lead to an understanding of the structural determinants, mechanism of potentiation, the role of GluN2C within the threshold of plasticity and in vivo effects of ketopyrrolines, a novel compound class that selectively potentiates the GluN1/GluN2C NMDA receptor.

Public Health Relevance

The goal of this project is to understand the mechanism of ketopyrrolines and the role of GluN2C on plasticity, a novel class of compounds that specifically potentiate the GluN1/GluN2C NMDA receptor. This research will provide insight into the structural components of the GluN1/GluN2C NMDA receptor and potentially the role of this receptor in synaptic plasticity. The data from this research could potentially be useful for developing new therapies for neurological disorders thought to be associated with aberrant NMDA receptor function such as, schizophrenia, autism, and psychosis.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F03B-A (20))
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Silberberg, Shai D
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Emory University
Schools of Medicine
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Zimmerman, Sommer S; Khatri, Alpa; Garnier-Amblard, Ethel C et al. (2014) Design, synthesis, and structure-activity relationship of a novel series of GluN2C-selective potentiators. J Med Chem 57:2334-56
Khatri, Alpa; Burger, Pieter B; Swanger, Sharon A et al. (2014) Structural determinants and mechanism of action of a GluN2C-selective NMDA receptor positive allosteric modulator. Mol Pharmacol 86:548-60