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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

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

Published: July 10th, 2018

DOI:

10.3791/58160

1Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor's biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

With current advances in medicine, life expectancy has increased significantly; however, so has the prevalence of age-related diseases. Diseases of the central nervous system (CNS) such as schizophrenia, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Parkinson's disease, among others, are no exception and have been projected to increase over the next decade1,2,3. The malfunction of ionotropic glutamate receptors known as N-methyl-D-aspartate receptors (NMDAR) has been linked to Alzheimer's disease, schizophrenia, traumatic brain injury, stroke, diab....

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1. Preparation of Cells

NOTE: This protocol, including data generation, uses HEK293 cells transduced with a baculovirus encoding NR1 and NR2A cells.

  1. Seed the appropriate number of HEK293 cells and add the NR1 and/or NR2A virus at the appropriate final concentrations (1.00 µL each). For a 384-well plate, use 10,000 cells/well in a final volume of 30 µL.
  2. Alternatively, seed HEK293-NR1-NR2A cells at the appropriate cell number and volume (for a 384-well plate, use .......

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Before testing the effects of small molecules, one must determine the optimal expression levels of NMDARs as well as the optimal ligand concentrations. As described, HEK293 cells were seeded at 10,000 cells per well in a 384-well plate, in the presence of 5 µM CGP060667, then transduced with varying amounts of NR1 and NR2A viruses. After incubation overnight, ligand-induced calcium flux was measured (Figure 1). The results of these experiments reveal the.......

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The success of this assay depends largely on the health of the HEK cells used. Cells undergoing exponential growth and with low passage number should be used. This assay involves many transfers and additions of solutions, so using caution will ensure higher accuracy in its results. Concentrations of compounds and all other reagents should be also cross-checked to minimize errors. When replacing cell media with the assay buffer for the calcium flux assay, the plate should be tilted gently so that the cells do not detach. .......

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The authors would like to thank the Post Baccalaureate Scholars Program Office and Novartis Institutes for BioMedical Research as a whole for funding this study.

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Name Company Catalog Number Comments
HEK-293 ATCC CRL-1573
Human NMDA (NR1/NR2A) Receptor Cell Line ChanTest Corporation CT6120
pFastBac Dual Expression Vector ThermoFisher Scientific 10712-024
Corning 384-well Clear Flat Bottom Microplate Corning Life Sciences 3844
FLIPR Calcium 6-QF Assay Kit Molecular Devices R8192
Glycine Sigma-Aldrich G7126
Glutamate Sigma-Aldrich 49621
D-serine Sigma-Aldrich S4250
L701,324 Tocris 907
HEPES Buffer Boston Bio Product BB-103
Magnesium Chloride Solution Sigma-Aldrich 63069
Calcium Chloride VWR E506
HBSS ThermoFisher Scientific 14025-092
Probenecid ThermoFisher Scientific P36400
DMEM/F-12, GlutaMAX media ThermoFisher Scientific 10565018
MDL105,519 NIBR Synthesized in house
NVP-AAM077 NIBR Synthesized in house
CGP070667 NIBR Synthesized in house

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