Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β(1a) Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes

The dihydropyridine receptor (DHPR) β(1a) subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β(1a) 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 β(1a) 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 β(1a) reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β(1a) 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 β(1a) 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 β(1a) -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β(1a) subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β(1a) and RyR1 in resting myotubes.