Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca2+ concentrations but inhibits it at micromolar Ca2+ concentrations, indicating that binding of Ca2+ to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca2+ sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (CaM)-Ca-ACR probe displayed high-affinity binding to, and Ca2+-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca2+, (CaM)-Ca-ACR exhibited a substantial (>50%) decrease in fluorescence (K-Ca = 2.7 +/- 0.8 muM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614-43)) caused an even more pronounced Ca2+-dependent fluorescence decrease, and a greater than or equal to10-fold leftward shift in its K-Ca (0.2 +/- 0.1 muM). In the presence of intact RyR1 channels in SR vesicles, (CaM)-Ca-ACR fluorescence spectra were distinct from those in the presence of RyR1(3614-43), although a Ca2+-dependent decrease in fluorescence was still observed. The K-Ca for (CaM)-Ca-ACR fluorescence in the presence of SR (0.8 +/- 0.4 muM) was greater than in the presence of RyR1(3614-43) but was consistent with functional determinations showing the conversion of (CaM)-Ca-ACR from channel activator (apoCaM) to inhibitor (Ca2+CaM) at Ca2+ concentrations between 0.3 and 1 muM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca2+ sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca2+ sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca2+.