ABSTRACT
Historically, the rst use of uorescence to study ion channel structure was to monitor the intrinsic uorescence of tryptophan and tyrosine (1,2). By exploiting intrinsic uorescence, no additional labeling technique was required, making these
experiments possible before the cloning of ion channels. However, using intrinsic uorescence of aromatic amino acids only succeeds if they do not occur too frequently in the protein and if they are involved in the specic process investigated. Otherwise, labeling has to be directed to the region of interest, which was rst reported with the synthesis of uorescently labeled bungarotoxin and tetrodotoxin derivatives (3,4). Fluorescence recordings of channel activity picked up signicantly with the development of uorescent calcium indicators in the early 1980s (5,6). Finally, with the cloning and recombinant expression of the voltage-gated sodium (7-9), Shaker potassium (10-12), and other ion channels (13-17), the molecular biology tools as well as the structural information became available to specically target important regions in the channels in order to follow their conformational changes. Accordingly, the rst voltage-clamp uorometry (VCF) measurements, simultaneous measurements of site-directed
Contents 9.1 Introduction 113 9.2 Modulation of uorescence 114
9.2.1 Solvent relaxation 115 9.2.2 Quenching 115 9.2.3 Förster resonance energy transfer 116
9.3 Obtaining structural information from uorescence measurements 117 9.3.1 Kinetics of local structural rearrangements 117
9.3.1.1 Intra-and intermolecular distance measurements 118 9.3.1.2 Measuring static distances with FRET 120 9.3.1.3 FRET using uorescence lifetime measurements 120 9.3.1.4 Lanthanide-based resonance energy transfer (LRET) 121 9.3.1.5 Resonance energy transfer in multimeric channels 122 9.3.1.6 Reconstructing atomistic models from FRET/LRET distances 123
9.3.2 Stoichiometry of multimeric channels 124 9.3.2.1 Fluorescence intensity measurements 124 9.3.2.2 Single subunit counting 124
9.3.3 Ligand binding 126 9.3.4 Single channel uorescence 126
9.4 Labeling techniques 127 9.4.1 iol-reactive chemistry 127 9.4.2 Fluorescently labeled ligands or toxins 127 9.4.3 Genetically encoded uorescent labels 127
9.4.3.1 Fluorescent proteins 127 9.4.3.2 Ligand-binding domains 128 9.4.3.3 Fluorescent unnatural amino acids 128
9.5 Conclusions 128 References 129
uorescence and electrophysiology, followed after just a few years (18,19) and were later extended to a wide variety of channels and other transport proteins. Single channel tracking in mammalian cells became possible by fusion with green uorescent protein (GFP) or one of the derivatives (20,21), and investigation of ion channels by uorescence spectroscopy was further improved by higher sensitivity down to the single molecule level and dierent labeling techniques.
