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Related Experiment Video

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A High-content Assay for Monitoring AMPA Receptor Trafficking
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Crosslinking glutamate receptor ion channels.

Andrew J R Plested1, Mette H Poulsen2

  • 1Institute of Biology, Cellular Biophysics, Humboldt Universität zu Berlin, Berlin, Germany; Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany; NeuroCure Cluster of Excellence, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Methods in Enzymology
|June 1, 2021
PubMed
Summary

This study presents a method for state-dependent crosslinking of glutamate-gated AMPA receptors using cysteine or unnatural amino acids (UAAs) in living cells. The approach enables millisecond-timescale analysis of ion channel movements and structural changes.

Keywords:
ElectrophysiologyFast perfusionPiezoUV lightUnnatural amino acids

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Area of Science:

  • Molecular biology
  • Neuroscience
  • Biophysics

Background:

  • Studying transient ion channel movements during gating is crucial for understanding cellular function.
  • Crosslinking strategies combined with other biochemical and biophysical methods offer powerful insights.
  • Glutamate-gated ion channels, particularly AMPA receptors, are key targets for such investigations due to their rapid activation.

Purpose of the Study:

  • To describe a protocol for state-dependent crosslinking of glutamate-gated AMPA receptors in living cells.
  • To enable millisecond-timescale analysis of ion channel conformational changes during gating.
  • To present strategies for analyzing crosslinking data computationally to understand structure-function relationships.

Main Methods:

  • Utilizing cysteine and photoactive unnatural amino acids (UAAs) for crosslinking in living cells.
  • Developing a perfusion tool for rapid chemical modification of AMPA receptors.
  • Employing electrophysiology, biochemistry, and in silico structural analysis.
  • Implementing light-driven crosslinking for receptors with UAA incorporation.

Main Results:

  • A protocol for rapid chemical modification and state-dependent crosslinking of AMPA receptors on the millisecond timescale was established.
  • Unnatural amino acids (UAAs) were successfully incorporated, although resulting in lower expression levels compared to cysteines.
  • Strategies were developed to capture light-driven oligomerization of UAA-containing AMPA receptors for biochemical analysis.
  • Computational methods were outlined for interpreting crosslinking data in structural and functional contexts.

Conclusions:

  • The described crosslinking approach, using either cysteines or UAAs, is effective for studying fast conformational dynamics of ion channels like AMPA receptors.
  • The developed system facilitates state-dependent crosslinking and structural analysis at millisecond resolution.
  • Computational strategies are essential for integrating crosslinking data to elucidate ion channel structure and function.