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

글리신/D-떠들고와 조미료에 감도와 NMDA 수용 체를 공부 하는 높은 처리량 칼슘-플럭스 분석 결과

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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

글리신/D-떠들고와 조미료에 감도와 NMDA 수용 체를 공부 하는 높은 처리량 칼슘-플럭스 분석 결과

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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.

이 프로토콜의 목표는 더 큰 규모에 NMDA 수용 체 (NMDAR)의 연구를 촉진 하기 위하여 작은 분자의 modulatory 효력의 검사 및 치료 응용 프로그램입니다.

이 방법은 신경 질환을 치료하기 위한 NMDA 조절제의 발견에 도움이 될 수 있습니다. 이 기술의 주요 장점은 방어 농도 또는 화합물의 리간드 조합에 대한 응답으로 NMDA 수용체 활성의 fissum 연구를 할 수 있다는 것입니다. 따라서 이 방법은 NMDA 수용체의 일반적인 기능에 대한 통찰력을 제공할 수 있습니다.

또한 리간드 또는 화합물에 대한 반응으로 생물학에 특이적인 아형과 NMDA 수용체의 민감도를 연구하는 데 적용할 수 있습니다. NMDA 수용체의 활성을 측정하기 위해 보호 화합물이 있는 상태에서 NR1, NR2 또는 HEK 293, NR1, NR2A 세포로 형질주입된 HEK 293 세포가 있는 384웰 플레이트를 준비합니다. 그런 다음 섭씨 37도에서 접시를 16시간 동안 5%의 이산화탄소로 배양합니다.

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