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High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

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Visualizing the triheteromeric N-methyl-D-aspartate receptor subunit composition.

Stephen Beesley¹, Akash Gunjan¹, Sanjay S Kumar¹

¹Department of Biomedical Sciences, College of Medicine and Program in Neuroscience, Florida State University, Tallahassee, FL, United States.

Frontiers in Synaptic Neuroscience

|June 9, 2023

Summary

Researchers visualized N-methyl-D-aspartate receptor (NMDAR) subunit composition at synapses using advanced microscopy. This confirms triheteromeric NMDARs and offers insights into neurodegenerative disease vulnerability.

Keywords:

GluN3 NMDA receptors confocal microscopy immunohistochemistry subunit composition t-NMDARs visualizing subunit composition

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Area of Science:

Neuroscience
Molecular Biology
Cell Biology

Background:

N-methyl-D-aspartate receptors (NMDARs) are crucial ligand-gated ionotropic channels mediating glutamate neurotransmission in the central nervous system.
Their Ca2+ influx capability links them to synaptic plasticity, cell death, and various neurological processes.
Subunit composition is traditionally inferred through cell biology, electrophysiology, and pharmacology.

Purpose of the Study:

To visualize synaptic NMDAR subunit composition directly in acute brain slices.
To confirm the presence of triheteromeric t-NMDARs and differentiate them from diheteromeric d-NMDARs.
To correlate NMDAR structure with function and identify potential mechanisms of excitotoxicity.

Main Methods:

Utilized highly specific antibodies targeting extracellular NMDAR subunit epitopes.
Employed high-resolution confocal microscopy for visualization in rat acute brain slices.
Co-imaged NMDARs with postsynaptic density (PSD-95) and presynaptic active zone (Bassoon) markers.

Main Results:

Successfully visualized synaptic NMDAR subunit composition with high precision.
Confirmed the expression of triheteromeric t-NMDARs (GluN1, GluN2, GluN3) at synapses for the first time.
Demonstrated co-localization of NMDARs with PSD-95, but not Bassoon, confirming synaptic localization.
Identified GluN3A-containing t-NMDARs as highly Ca2+ permeable and linked to excitotoxicity.

Conclusions:

Direct visualization of NMDAR subunit composition is achievable using antibody-based super-resolution microscopy.
This technique confirms the existence and synaptic localization of triheteromeric NMDARs, reconciling functional data.
Findings highlight the role of GluN3A-containing NMDARs in excitotoxicity and suggest potential applications for neurodegenerative disease research, such as Temporal Lobe Epilepsy.