Cell Reports
Volume 22, Issue 6, 6 February 2018, Pages 1615-1626
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High-Sensitivity Fluorometry to Resolve Ion Channel Conformational Dynamics

https://doi.org/10.1016/j.celrep.2018.01.029Get rights and content
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Highlights

  • Increased fluorescence sensitivity and low background

  • Up to 10 kHz fluorescence time resolution

  • High versatility regarding fluorescent dyes and membrane proteins

  • Mechanistic insights into voltage- and ligand-gated ion channels

Summary

Fluorescent labels offer the capability to follow conformational dynamics of membrane proteins, but signal detection in such recordings is inherently difficult to achieve in a cell membrane and lacks sufficient time resolution to follow physiologically relevant transitions. Here, we develop high-sensitivity patch-clamp fluorometry (hsPCF), a fluorescence-based approach that results in up to 10-fold increased signals and affords 50-fold faster fluorescence recordings than previous methods. The increased time resolution is paired with a very high versatility in terms of the choice of fluorescent dye, cell type, and protein of interest. We highlight this versatility by providing insight into the conformational dynamics of both ligand- and voltage-gated ion channels using fluorescent labels introduced in extracellular or transmembrane positions while changing either the extra- or intracellular solutions. Together, hsPCF will thus enable the future study of membrane-embedded proteins with sufficient temporal resolution to resolve conformational dynamics.

Keywords

protein conformations
glycine receptor
acid-sensing ion channel
Shaker potassium channel
noncanonical amino acids
unnatural amino acids

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