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 Ca ²⁺ 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 ACRCaM probe displayed high-affinity binding to, and Ca²⁺-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca ²⁺, ^ACRCaM exhibited a substantial (> 50%) decrease in fluorescence (K_Ca = 2.7 ± 0.8 μM). A peptide derived from the RyR1 CaM binding domain (RyR1_3614-43) caused an even more pronounced Ca^2--dependent fluorescence decrease, and a ≥ 10-fold leftward shift in its K_Ca (0.2 ± 0.1 μM). In the presence of intact RyR1 channels in SR vesicles, ACRCaM 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 ^ACRCaM fluorescence in the presence of SR (0.8 ± 0.4 μM) was greater than in the presence of RyR1_3614-43 but was consistent with functional determinations showing the conversion of ^ACRCaM from channel activator (apoCaM) to inhibitor (Ca ²⁺CaM) at Ca²⁺ concentrations between 0.3 and 1 μM. 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⁺.