The goal of this project is to define the molecular mechanism underlying subtype-specificity in N-methyl-D- aspartate receptors (NMDARs) to facilitate development of subtype-specific reagents for controlling NMDAR activities. NMDARs belong to the family of ionotropic glutamate receptors, which mediate the majority of fast excitatory synaptic transmission in mammalian brains. Abnormal activity of NMDARs is implicated in various neurological disorders and diseases including schizophrenia, depression, Alzheimer's disease, and Parkinson's disease. Those receptors are multimeric ligand-gated ion channels composed mainly of GluN1 and GluN2 subunits that bind to glycine and L-glutamate at the extracellular domain, respectively. Gating of transmembrane ion channels is mediated by concurrent binding of glycine and L-glutamate to the ligand- binding domain (LBD) and is allosterically regulated by binding of modulator compounds including phenylethanolamines and Zn2+ to the amino terminal domain (ATD). Importantly, functional properties of NMDARs subtypes, which are defined by four distinct GluN2 subunits (A though D), exhibit dramatically different functional properties. Different NMDAR subtypes are expressed in discrete regions of the brain at given developmental stages and are also associated with distinct neurological diseases and disorders. Thus, understanding the molecular basis for subtype-specificity will be necessary in order to develop specific reagents for treatment of the above neurological diseases. Despite much enthusiasm, the field is limited to one useful subtype-specific compound, phenylethanolamine, which targets GluN1/GluN2B NMDARs but is associated with off-target effects when used therapeutically. Development of reagents targeting other subtypes such as GluN1/GluN2A has been hampered due to limited amount of structural information on subtypes of NMDARs, which would allow comprehensive structural comparison. To obtain a mechanistic understanding of subtype-specificity in NMDARs and to facilitate development of subtype-specific reagents for GluN1/GluN2A and GluN1/GluN2B NMDARs, we will conduct research aimed at:
Aim 1 obtaining an in-depth understanding of the ligand-binding site in GluN1/GluN2B ATD and GluN1/GluN2A LBD;
Aim 2 defining the molecular mechanism of subtype-specific allosteric inhibition in GluN1/GluN2A ATD;
and Aim 3 determining the binding and inhibition mechanism of GluN1/GluN2B NMDAR by inhibitory antibody that we recently developed. These three goals will be achieved by obtaining the structural information of ATD and LBD and testing structure- based hypotheses by electrophysiology. Successful completion of the proposed studies will provide unprecedented insights into ligand-binding sites in ATD, LBD, and molecular elements underlying subtype- specificity, and to demonstrate a novel approach to inhibit NMDAR in a subtype-specific manner using inhibitory antibodies. These findings will facilitate development of subtype-specific reagents to study and treat the mental health related disorders above.

Public Health Relevance

The proposed studies aim to determine the molecular basis for subtype-specificity in N-methyl-D- aspartate receptor (NMDAR) ion channels, which are critically involved in brain function and development. The studies are relevant to public health since abnormal NMDAR activities are implicated in various brain disorders and diseases including depression, schizophrenia, and Alzheimer's diseases. In-depth understanding of NMDAR structures will pinpoint molecular determinants for subtype-specificity and facilitate development of specific reagents with therapeutic values to treat the above diseases and disorders.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Research Project (R01)
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Biophysics of Neural Systems Study Section (BPNS)
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Nadler, Laurie S
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Cold Spring Harbor Laboratory
Cold Spring Harbor
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Regan, Michael C; Grant, Timothy; McDaniel, Miranda J et al. (2018) Structural Mechanism of Functional Modulation by Gene Splicing in NMDA Receptors. Neuron 98:521-529.e3
Amin, Johansen B; Salussolia, Catherine L; Chan, Kelvin et al. (2017) Divergent roles of a peripheral transmembrane segment in AMPA and NMDA receptors. J Gen Physiol 149:661-680
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Regan, Michael C; Romero-Hernandez, Annabel; Furukawa, Hiro (2015) A structural biology perspective on NMDA receptor pharmacology and function. Curr Opin Struct Biol 33:68-75
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