Structural Dynamics of Calmodulin in Regulation of Calcium Release in Health and Disease

Abstract

We have used time-resolved fluorescence (FRET) and EPR (DEER) spectroscopies to study the structural changes in calmodulin (CaM) that are relevant to regulation of the muscle calcium release channel, the ryanodine receptor (RyR). Regulation of RyR by CaM is disrupted by oxidation and disease-causing mutations. However, the structural basis for these regulatory changes, and the role CaM plays in the development of heart failure and arrhythmias, is not well understood. Several studies suggest that the modulatory role of CaM is closely tied to its conformation when bound to RyR, but the correlation between structure and function in physiologically relevant conditions is largely unknown. To test the hypothesis that the modulatory action of CaM on RyR is caused by structural changes in the CaM-RyR complex, we use site-directed spectroscopy to determine the structural changes that contribute to calcium regulation in skeletal and cardiac muscle. The approach is to prepare CaM mutants that contain a single Cys on each of the two lobes (N and C), then attach spectroscopic probes to those sites. Ryanodine binding measurements are performed to ensure functional integrity of labeled CaM constructs. Then, we measure changes in intra-CaM distance distributions using time-resolved FRET or DEER. Our previous studies employed DEER of isolated CaM. Now, we rely on time-resolved FRET, to resolve structural changes of CaM bound to functional RyR in sarcoplasmic reticulum membranes. We will compare these measurements with those of CaM bound to a peptide corresponding to the CaM-binding domain of RyR (RyR1 residues: 3614-3643). This work was supported by NIH grants AG26160 and HL092097 (to DDT) and a Predoctoral Fellowship from the American Heart Association 15PRE25700131 (to MRM).