title: Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.
abstract: The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable. We used fluorescence resonance energy transfer (FRET) to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP) was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1) myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.
authors.affiliation: Cellular and Molecular Arrhythmia Research Program, Department of Medicine, University of Wisconsin-Madison, Wisconsin, United States of America.
authors.forename: Dipankar
authors.initials: D
authors.lastname: Bhattacharya
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Wisconsin, United States of America.
authors.forename: Andrew
authors.initials: A
authors.lastname: Mehle
authors.affiliation: Cellular and Molecular Arrhythmia Research Program, Department of Medicine, University of Wisconsin-Madison, Wisconsin, United States of America.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kamp
authors.affiliation: Cellular and Molecular Arrhythmia Research Program, Department of Medicine, University of Wisconsin-Madison, Wisconsin, United States of America.
authors.forename: Ravi C
authors.initials: RC
authors.lastname: Balijepalli
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D051379:Mice; D008817:Mice, Mutant Strains; D018485:Muscle Fibers, Skeletal; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*
chemical_list: D020746:Calcium Channels, L-Type; D019837:Ryanodine Receptor Calcium Release Channel; C000592747:ryanodine receptor 1, mouse