# Subsynaptic organization of the major NMDAR subtypes

> **NIH NIH F31** · UNIVERSITY OF MARYLAND BALTIMORE · 2021 · $41,935

## Abstract

Glutamatergic signaling via NMDA receptors (NMDARs) is critical in synaptic plasticity, excitotoxicity, and
numerous degenerative and cognitive disorders. Each NMDAR comprises four subunits: two obligatory GluN1
subunits and two GluN2A-D subunits. Notably, NMDARs with different subunit compositions display different
channel kinetics and protein interactions, supporting the widely held notion that subunit composition dictates
NMDAR function. The subsynaptic organization of NMDARs influences the probability of their activation during
synaptic transmission, and extrasynaptic receptors are thought to play roles in cell death, plasticity and gene
expression. However, despite evidence for subunit-specific NMDAR nanoclustering, to date there is almost no
information about the nanoscale distribution of specific NMDAR subunits within synapses or with respect to sites
of vesicular release. Thus, to elucidate the organization of synaptic and extrasynaptic NMDARs with respect to
key postsynaptic molecules and presynaptic release sites, in the first aim of my project, I will use a combination
of CRISPR-mediated gene tagging and nanobody labeling with DNA-Exchange PAINT super-resolution imaging.
These measures will provide a comprehensive map of major NMDAR subunits and provide insight into their roles
in neurons. Next, though many NMDARs contain GluN1 and two identical GluN2 subunits, in many brain regions,
NMDARs commonly are “triheteromeric” receptors containing both GluN2A and GluN2B with GluN1. However,
despite their deduced importance, there are no methods to unambiguously distinguish triheteromeric NMDARs
from other subtypes in neurons, and thus their distribution and subcellular trafficking remain mysterious. To
overcome this, I have designed a method for direct visualization of triheteromeric NMDARs using bimolecular
fluorescent complementation (BiFC). I tagged GluN2A and GluN2B with two parts of a modified fluorescent
protein that complement to produce fluorescence only when an NMDAR is assembled containing both the
GluN2A and GluN2B subunits (split-tagged NMDARs). In the second aim of my proposal, following a series of
validation experiments, I will use these split-tagged NMDARs to address two straightforward but longstanding
questions surrounding NMDARs. First, though GluN2A and GluN2B traffic with different dynamics and target
subsynaptic regions in part through differing interactions with scaffolding molecules, it is unknown which subunit
dominates synaptic integration of triheteromeric receptors. Using my new probes, I will provide the first direct
measure of triheteromeric synaptic enrichment. Second, the rate of NMDAR turnover in synapses is critical for
determining synaptic strength and plasticity, yet the exchange rate and mobile fraction of triheteromeric receptors
have not previously been possible to measure. Using FRAP, I will time determine if triheteromeric NMDARs
display unique turnover compared to other NMDARs. Completion of this work...

## Key facts

- **NIH application ID:** 10232435
- **Project number:** 1F31MH124283-01A1
- **Recipient organization:** UNIVERSITY OF MARYLAND BALTIMORE
- **Principal Investigator:** Michael C. Anderson
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $41,935
- **Award type:** 1
- **Project period:** 2021-04-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10232435

## Citation

> US National Institutes of Health, RePORTER application 10232435, Subsynaptic organization of the major NMDAR subtypes (1F31MH124283-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10232435. Licensed CC0.

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