Structural Dynamics Of Calmodulin In Regulation Of Ryr Calcium Release Channels

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study the structural changes in calmodulin (CaM) that are relevant to regulation of the skeletal muscle calcium release channel, the ryanodine receptor (RyR1 isoform). 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 RyR1 is caused by structural changes in the CaM-RyR1 complex, we used TR-FRET between two fluorescent probes covalently bound to each lobe (N and C) of CaM to measure Ca2+-dependent changes in the distance between lobes when CaM is bound to RyR1 in sarcoplasmic reticulum membranes, isolated RyR1, and a peptide corresponding to a CaM-binding domain of RyR1 (RyRp; RyR residues 3614-3643). [3H]-ryanodine binding measurements were performed to ensure functional integrity of labeled CaM constructs, and to directly correlate the FRET structural readout with the CaM-RyR functional interaction. At both low and high Ca2+, we find that CaM adopts a unique structure when bound to full-length RyR1 compared with binding to RyRp. These data suggest that the structure of CaM when bound to the peptide is not representative of CaM binding to full-length RyR1. This striking result has implications for many other CaM binding partners that have been structurally characterized through peptide studies. Ongoing studies utilize purified RyR1 to directly measure CaM-RyR binding kinetics as well as utilize this CaM biosensor to determine the structural changes that contribute to regulation of the cardiac RyR isoform (RyR2). This work was supported by NIH grants AG26160 (DDT), HL092097 (RLC) and 1F31AG052329-01A1 (MRM) and an American Heart Association Predoctoral Fellowship 15PRE25700131 (MRM).