A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

Fred Yeboah; Hongqiu Guo; Anke Bill

doi:10.3791/58160

NMDA reseptörleri glisin/D-serin ve glutamat duyarlılık ile çalışmak için bir yüksek-den geçerek ...

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Neuroscience

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JoVE Journal Neuroscience

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

NMDA reseptörleri glisin/D-serin ve glutamat duyarlılık ile çalışmak için bir yüksek-den geçerek kalsiyum-flux tahlil

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04:48 min

July 10, 2018

DOI: 10.3791/58160-v

Fred Yeboah¹, Hongqiu Guo¹, Anke Bill¹

¹Chemical Biology and Therapeutics,Novartis Institutes for BioMedical Research

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Overview

This study presents a protocol aimed at investigating NMDA-receptors (NMDAR) to explore the modulatory effects of small molecules, focusing on their therapeutic applications for neurological diseases. The protocol utilizes HEK 293 cells transduced with NMDAR subunits for a detailed analysis of receptor activity.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Pharmacology

Background

NMDA-receptors play a crucial role in synaptic plasticity and memory function.
Modulation of these receptors can lead to novel treatments for neurological disorders.
The protocol allows high-throughput analysis of receptor activity in response to different ligands.

Purpose of Study

To facilitate large-scale studies of NMDA-receptor activity.
To assess the effects of small molecules on receptor modulation.
To enhance understanding of NMDA receptor pharmacology.

Methods Used

The key platform used is HEK 293 cell culture, specifically modified to express NMDAR subunits NR1 and NR2A.
Cells are treated with compounds and ligands, followed by fluorescence measurements to analyze receptor activity.
Incubation and washing steps are executed to optimize the assay conditions.
Calcium-flux is measured using a fluorescence dye to determine receptor responsiveness.

Main Results

The method allows characterization of specific NMDA receptor subtypes in response to various ligands and antagonists.
Significant findings include the examination of the glutamate binding site antagonist NVP-AAM077 and glycine binding site antagonist L701, 324.
The results facilitate a better understanding of NMDA receptor modulation and its implications for therapeutic development.

Conclusions

This study enables the exploration of NMDAR dynamics and their modulation by small molecules.
It highlights the potential for drug development targeting NMDA receptor-related neurological conditions.
The findings contribute to our understanding of receptor pharmacology and associated therapeutic strategies.

Frequently Asked Questions

What are the advantages of using HEK 293 cells for studying NMDA receptors?

HEK 293 cells are easily transfected, allowing for the expression of NMDA receptor subunits. They provide a controlled environment for high-throughput screening of compounds.

How is the NMDA receptor activity measured in this study?

The activity is measured using a calcium-flux assay, where fluorescence changes indicate receptor responsiveness to ligands and antagonists.

What types of data are obtained from the calcium-flux assay?

Data obtained includes baseline and maximal fluorescence ratios, which quantify NMDA receptor activity and response to compounds.

Can this method be applied to study other receptor subtypes?

Yes, the method can be adapted to investigate various receptor subtypes by using different combinations of ligands and cell lines.

What considerations should be taken when preparing cell cultures?

It is important to use non-confluent, healthy cells to ensure optimal receptor expression and response during assays.

What limitations should be kept in mind when using this protocol?

Care should be taken in the concentrations of compounds and ligands used, as well as managing the timing of incubations to maintain assay integrity.

NMDA reseptörleri (NMDAR) çalışma daha büyük ölçekli, kolaylaştırmak ve küçük moleküller düzenleyici etkileri muayene ve tedavi uygulamaları izin vermek için bu iletişim kuralını hedefidir.

Bu yöntem, nörolojik hastalıkları tedavi etmek için NMDA modülatörlerinin keşfine yardımcı olabilir. Bu tekniğin temel avantajı, bileşiklerdeki savunma konsantrasyonlarına veya ligandların kombinasyonlarına yanıt olarak NMDA-reseptör aktivitesinin fissum çalışmasına izin vermesidir. Dolayısıyla bu yöntem, NMDA reseptörünün genel kapasitesi hakkında fikir verebilir.

Ligandlara veya bileşiklere yanıt olarak biyolojiye özgü alt tipleri ve NMDA reseptörünün duyarlılığını incelemek için de uygulanabilir. NMDA reseptörünün aktivitesini ölçmek için, koruyucu bileşik varlığında NR1, NR2 veya HEK 293, NR1, NR2A hücreleri ile transdüksiyona uğrayan HEK 293 hücreli bir 384 kuyulu plaka hazırlayın. Ardından, plakayı 37 santigrat derecede ve 16 saat boyunca yüzde beş karbondioksitte inkübe edin.

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