# Structure and function of hetero-multimeric ligand-gated ion channels

> **NIH NIH R01** · COLD SPRING HARBOR LABORATORY · 2021 · $575,545

## Abstract

PROJECT SUMMARY
The goal of this project is to gain an in-depth mechanistic understanding about multi-heteromeric ion channels,
N-methyl-D-aspartate receptors (NMDARs), regulated by different compounds, splice variants, and subtypes.
These receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of
excitatory synaptic transmission and play significant roles in basic brain functions, development, and neurological
disorders such as seizures, strokes, depression, and schizophrenia, as well as Parkinson’s and Alzheimer’s
diseases. NMDARs are hetero-multimeric ligand-gated ion channels composed of GluN1 and GluN2 and/or
GluN3 subunits. The GluN1 and GluN3 subunits bind co-agonists including glycine and D-serine, whereas the
GluN2 subunits bind the neurotransmitter, glutamate. Each subunit protein is composed of an amino terminal
domain (ATD), a ligand-binding domain (LBD), a transmembrane domain (TMD), and a carboxyl terminal domain
(CTD). The GluN1-GluN2 NMDARs open their transmembrane ion channels upon binding of glycine and
glutamate, whereas the GluN1-GluN3 NMDARs activate by glycine alone. We recently solved the first structure
of the intact hetero-tetrameric rat GluN1-GluN2B NMDARs, initially by x-ray crystallography and later by cryo-
electron microscopy. Despite recent advances, there are many fundamental questions remaining regarding the
mechanism of activation, desensitization, inhibition by competitive antagonists, and functional diversity elicited
by alternative splicing in GluN1 and different subunit combinations (GluN1-GluN2A-D and GluN3A-B). The
structure of the GluN3 NMDARs is limited to that of the GluN3A LBD, thus, restricting our understanding about
how this under-studied subtype form ion channels and mediate functions. Thus, the goal of the proposed project
is to investigate patterns of compound bindings, protein conformations and subunit arrangements in various
functional states and subtypes of NMDARs. To achieve these goals, we will conduct research aimed at: Aim 1
determining the mechanisms of activation, desensitization, and inhibition by agonists and antagonists; Aim 2
defining the underlying mechanism for altered pH-sensitivity and deactivation speeds by polyamines in a splice-
variant-specific manner; and Aim 3 revealing the architecture of the GluN1-GluN3 NMDAR for the first time.
These three aims will be achieved by obtaining the structural information of the GluN1-GluN2B NMDAR in the
presence of agonists and antagonists, splice variants with and without spermine, and the GluN1-GluN3A
NMDAR by a combination of x-ray crystallography and cryo-EM. The structure-based functional hypotheses will
be tested by experiments involving electrophysiology. Successful completion of the proposed studies will provide
in-depth information into the sophisticated function of NMDAR subtypes and splice variants mediated by
compound bindings followed by rearrangement of multiple subunits and domains. T...

## Key facts

- **NIH application ID:** 10128516
- **Project number:** 5R01NS111745-03
- **Recipient organization:** COLD SPRING HARBOR LABORATORY
- **Principal Investigator:** Hiroyasu Furukawa
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $575,545
- **Award type:** 5
- **Project period:** 2019-04-15 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10128516

## Citation

> US National Institutes of Health, RePORTER application 10128516, Structure and function of hetero-multimeric ligand-gated ion channels (5R01NS111745-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10128516. Licensed CC0.

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