Methods for labeling skeletal muscle ion channels site-specifically with fluorophores suitable for FRET-based...

Skeletal muscle excitation-contraction coupling is triggered by the concerted action of two enormous Ca(2+) channel complexes, the dihydropyridine receptor and the type 1 ryanodine receptor. Recent advances in our understanding of the structure of these large Ca(2+) channels have been driven by fluorescence resonance energy transfer (FRET)-based analysis. A methodological challenge in conducting these FRET measurements is the ability to site-specifically label these huge ion channels with donor and acceptor fluorophores capable of undergoing energy transfer. In this chapter, we detail specific protocols for tagging large membrane proteins with these fluorescent probes using three orthogonal labeling methods: fluorescent protein fusions, biarsenical reagents directed to engineered tetracysteine tags, and Cy3/5 nitrilotriacetic acid conjugates that bind to poly-histidine tags.