title: Methods for labeling skeletal muscle ion channels site-specifically with fluorophores suitable for FRET-based structural analysis.
abstract: 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.
authors: Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.
authors: Mohana
authors: M
authors: Mahalingam
authors: Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA. Electronic address: jfessenden@partners.org.
authors: James D
authors: JD
authors: Fessenden
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000032:analysis* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis* / Q000378:metabolism
chemical_list: D020746:Calcium Channels, L-Type; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D049452:Green Fluorescent Proteins; C033223:polyhistidine; D006639:Histidine
keywords: Calcium; Cy3NTA; DHPR; EC coupling; FRET; FlAsH; Fluorescent protein; His tag; ReAsH; RyR1; Skeletal muscle