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Updated: Feb 20, 2026

Monitoring Leucine-Rich Repeat Containing 8 Channel (LRRC8/VRAC) Activity Using Sensitized-Emission Förster Resonance Energy Transfer (SE-FRET)
Published on: August 9, 2024
Studying Structural Dynamics of Potassium Channels by Single-Molecule FRET
Shizhen Wang1, Joshua B Brettmann2, Colin G Nichols3
1Center for the Investigation of Membrane Excitability Diseases and Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, 63110, USA. shizhen.wang@wustl.edu.
Abstract:
Single-molecule FRET (smFRET) can visualize conformational dynamics of individual ion channels in lipid bilayers of defined composition. Dynamic and distance measurements from smFRET, combined with single channel recordings, can provide previously unattainable direct mechanistic insights into ion channel function and modulation. smFRET measurements require site-specific fluorophore labeling between two distinct sites, which is a major challenge for multimeric ion channels. This chapter aims to provide a step-by-step protocol: (1) to design concatemeric constructs with only two cysteine residues within a homotetrameric channel; (2) to express, purify, label, and reconstitute channel proteins; (3) to perform smFRET imaging on channel proteins in liposomes with an objective-based Total Internal Reflection (TIRF) microscope; and finally (4) to analyze the FRET distributions and dynamics that reflect the dynamic conformational transitions of ion channels in membranes.
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