The overall goal of the research studies proposed here is to obtain high-resolution structures of intact hetero- multimeric N-methyl-D-aspartate receptors (NMDARs). NMDARs belong to the family of ionotropic glutamate receptors, which mediate the majority of excitatory synaptic transmission in mammalian brains. Dysfunctional NMDARs are implicated in various neurological disorders and diseases including schizophrenia, depression, Alzheimer's disease, and Parkinson's disease. A unique aspect of NMDARs is that they are obligatory hetero- tetramers or higher oligomers composed of GluN1 and GluN2 (A-D) or GluN3 (A-B) subunits. Opening of NMDAR ion channels requires binding of glycine to GluN1 and GluN3 and glutamate to GluN2. To date, structural studies of NMDARs have been limited to the hetero-dimeric structures of the GluN1 and GluN2 extracellular domains. Thus, there is no clear knowledge on how subunits and domains are arranged to form hetero-multimeric ion channels and how transmembrane ion channel pores are shaped to confer specific properties of NMDAR ion channels including high calcium conductance and voltage-dependent magnesium block. Despite various technological breakthroughs, success in crystallographic studies on eukaryotic membrane proteins has been limited due to difficulties in expression, purification, and crystallization stemming from sample heterogeneity and instability. Importantly, there has been no crystal structure of eukaryotic hetero- multimeric membrane proteins that are recombinantly produced to date. The fact that numerous ion channels, G protein-coupled receptors, receptor kinases, and intramembrane proteases implicated in neurological diseases exist as hetero-multimers in native states points to the great need for structural studies on hetero- multimeric membrane proteins. To obtain the first crystal structure of hetero-multimeric ion channels and to understand the structure-function relationship of NMDARs, we will conduct research with the following two aims:
Aim 1 is to produce intact hetero-multimeric NMDAR proteins using our novel methodology and to biochemical characterize the homogeneously purified proteins;
and Aim 2 is to complete structural analysis of intact NMDARs in complex with various ligands reflecting different functional states by applying cutting-edge techniques in membrane protein crystallography and validate structure-based functional hypotheses by biochemical and electrophysiological experiments. Successful completion of the proposed studies is expected to result in the first crystal structure of a hetero-multimeric ion channel and to provide a mechanistic understanding of NMDARs that are critical in brain physiology and development. Importantly, the structural information obtained here will also provide strategies to develop compounds with therapeutic efficacy in neurological disorders and diseases. Furthermore, these studies on NMDARs will establish fundamental guidelines for crystallography on hetero-multimeric membrane proteins.

Public Health Relevance

The proposed studies aim to uncover the molecular basis for the structure and function of intact hetero- multimeric NMDA receptor ion channels, which are crucial in normal brain function and development. The studies are relevant to public health because dysfunctional NMDA receptors are implicated in various neurological disorders and diseases including seizure, stroke, schizophrenia, as well as Parkinson's and Alzheimer's diseases. Defining the molecular structure of the intact NMDA receptor is expected to unravel the structural elements important for basic functions and support designing of compounds that are useful in treating the above diseases and disorders.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biophysics of Neural Systems Study Section (BPNS)
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Chin, Jean
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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
Romero-Hernandez, Annabel; Furukawa, Hiro (2017) Novel Mode of Antagonist Binding in NMDA Receptors Revealed by the Crystal Structure of the GluN1-GluN2A Ligand-Binding Domain Complexed to NVP-AAM077. Mol Pharmacol 92:22-29
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Malinauskas, Tomas; Furukawa, Hiro (2016) Production of Heteromeric Transmembrane Receptors with Defined Subunit Stoichiometry. Structure 24:653-655
Regan, Michael C; Furukawa, Hiro (2016) Deeper Insights into the Allosteric Modulation of Ionotropic Glutamate Receptors. Neuron 91:1187-1189
Tajima, Nami; Karakas, Erkan; Grant, Timothy et al. (2016) Activation of NMDA receptors and the mechanism of inhibition by ifenprodil. Nature 534:63-8
Romero-Hernandez, Annabel; Simorowski, Noriko; Karakas, Erkan et al. (2016) Molecular Basis for Subtype Specificity and High-Affinity Zinc Inhibition in the GluN1-GluN2A NMDA Receptor Amino-Terminal Domain. Neuron 92:1324-1336
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
Karakas, Erkan; Regan, Michael C; Furukawa, Hiro (2015) Emerging structural insights into the function of ionotropic glutamate receptors. Trends Biochem Sci 40:328-37

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