Klinger M, Bofill-Cardona E, Mayer B, Nanoff C, Freissmuth M, Hohenegger M
Institute of Pharmacology, University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria.
Biochem J. 2001 May 1;355(Pt 3):827-33. doi: 10.1042/bj3550827.
Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin. Suramin and its analogue NF307 inhibit the interaction of calmodulin with the ryanodine receptor. We have investigated whether inhibition of calmodulin binding to target proteins is a general property of these compounds. Suramin inhibited binding of [(125)I]calmodulin to porcine brain membranes and to sarcoplasmic reticulum from skeletal muscle (IC(50)=4.9+/-1.2 microM and 19.9+/-1.8 microM, respectively) and blocked the cross-linking of [(125)I]calmodulin to some, but not all, target proteins in brain membranes by [(125)I]calmodulin. Four calmodulin-binding proteins were purified [ryanodine receptor-1 (RyR1) from rabbit skeletal muscle, neuronal NO synthase (nNOS) from Sf9 cells, G-protein betagamma dimers (Gbetagamma) from porcine brain and a glutathione S-transferase-fusion protein comprising the C-terminal calmodulin-binding domain of the metabotropic glutamate receptor 7A (GST-CmGluR7A) from bacterial lysates]. Three of the proteins employed (Gbetagamma, GST-CmGluR7A and RyR1) display a comparable affinity for calmodulin (in the range of 50-70 nM). Nevertheless, suramin and NF307 only blocked the binding of Gbetagamma and RyR1 to calmodulin-Sepharose. In contrast, the association of GST-CmGluR7A and nNOS was not impaired, whereas excess calmodulin uniformly displaced all proteins from the matrix. Thus suramin and NF307 are prototypes of a new class of calmodulin antagonists that do not interact directly with calmodulin but with calmodulin-recognition sites. In addition, these compounds discriminate among calmodulin-binding motifs.
靶蛋白上的钙调蛋白结合位点在一级序列上表现出相当大的差异；因此，与使钙调蛋白失活的化合物相比，阻断钙调蛋白与这些结合位点结合的化合物可能具有更高的选择性。苏拉明及其类似物NF307可抑制钙调蛋白与兰尼碱受体的相互作用。我们研究了抑制钙调蛋白与靶蛋白的结合是否是这些化合物的普遍特性。苏拉明抑制[¹²⁵I]钙调蛋白与猪脑膜以及骨骼肌肌浆网的结合（IC₅₀分别为4.9±1.2微摩尔/升和19.9±1.8微摩尔/升），并通过[¹²⁵I]钙调蛋白阻断[¹²⁵I]钙调蛋白与脑膜中部分而非全部靶蛋白的交联。纯化了四种钙调蛋白结合蛋白[兔骨骼肌中的兰尼碱受体-1（RyR1）、Sf9细胞中的神经元型一氧化氮合酶（nNOS）、猪脑膜中的G蛋白βγ二聚体（Gβγ）以及细菌裂解物中包含代谢型谷氨酸受体7A（GST-CmGluR7A）C末端钙调蛋白结合结构域的谷胱甘肽S-转移酶融合蛋白]。所使用的三种蛋白（Gβγ、GST-CmGluR7A和RyR1）对钙调蛋白表现出相当的亲和力（在50 - 70纳摩尔范围内）。然而，苏拉明和NF307仅阻断Gβγ和RyR1与钙调蛋白琼脂糖的结合。相反，GST-CmGluR7A和nNOS的结合并未受到损害，而过量的钙调蛋白能将所有蛋白从基质上均匀置换下来。因此，苏拉明和NF307是一类新型钙调蛋白拮抗剂的原型，它们不直接与钙调蛋白相互作用，而是与钙调蛋白识别位点相互作用。此外，这些化合物能区分不同的钙调蛋白结合基序。
