Fruen Bradley R, Balog Edward M, Schafer Janet, Nitu Florentin R, Thomas David D, Cornea Razvan L
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Biochemistry. 2005 Jan 11;44(1):278-84. doi: 10.1021/bi048246u.
Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) 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 Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).
钙调蛋白（CaM）在纳摩尔浓度的Ca²⁺条件下激活骨骼肌兰尼碱受体（RyR1），但在微摩尔浓度的Ca²⁺条件下抑制它，这表明Ca²⁺与CaM的结合可能为调节RyR1通道活性提供一个分子开关。为了直接检测与RyR1复合的CaM对Ca²⁺的敏感性，我们使用了一种对环境敏感的CaM丙烯罗丹加合物。得到的（ACR）CaM探针显示出与未标记的野生型（WT）CaM相似的对RyR1的高亲和力结合以及Ca²⁺依赖性调节。加入Ca²⁺后，（ACR）CaM的荧光显著降低（>50%）（KCa = 2.7±0.8微摩尔）。一种源自RyR1 CaM结合结构域的肽（RyR1(3614)(-)(43)）引起更明显的Ca²⁺依赖性荧光降低，并且其KCa向左移动≥10倍（0.2±0.1微摩尔）。在肌浆网（SR）囊泡中存在完整的RyR1通道时，（ACR）CaM荧光光谱与存在RyR1(3614)(-)(43)时不同，尽管仍观察到Ca²⁺依赖性荧光降低。在存在SR的情况下（ACR）CaM荧光的KCa（0.8±0.4微摩尔）大于存在RyR1(3614)(-)(43)时，但与功能测定结果一致，该结果表明在0.3至1微摩尔的Ca²⁺浓度下（ACR）CaM从通道激活剂（脱钙CaM）转变为抑制剂（Ca²⁺CaM）。这些结果表明与RyR1靶点的结合引起CaM结构和Ca²⁺敏感性的显著变化（即CaM调节）。然而，CaM与全长四聚体通道结合所导致的变化明显不同于由RyR1衍生肽引起的变化。我们认为与全长通道复合时CaM的Ca²⁺敏感性可能被调节以响应Ca²⁺的生理相关变化。
