- The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca2+ release during excitation–contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators.
- The Ca-ATPase isoform 2a (SERCA2a) pumps cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. Abnormally high cytosolic [Ca2+] is a central factor in heart failure, suggesting that augmentation of SERCA2a Ca2+ transport activity could be a promising therapeutic approach. SERCA2a is inhibited by the protein phospholamban (PLB), and a novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing PLB from binding to SERCA2a.
- S100A1 has been suggested as a therapeutic agent to enhance myocyte Ca2+ cycling in heart failure, but its molecular mode of action is poorly understood. Using FRET, we tested the hypothesis that S100A1 directly competes with calmodulin (CaM) for binding to intact, functional ryanodine receptors type I (RyR1) and II (RyR2) from skeletal and cardiac muscle, respectively. Our FRET readout provides an index of acceptor-labeled CaM binding near donor-labeled FKBP (FK506-binding protein 12.6) on the cytoplasmic domain of RyR in isolated sarcoplasmic reticulum vesicles.