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: Shaker potassium channel; acid-sensing ion channel; glycine receptor; noncanonical amino acids; protein conformations; unnatural amino acids.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.