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首页> 外文期刊>Biochemistry >Calcium Binding to Calmodulin Mutants Having Domain-Specific Effects on the Regulation of Ion Channels
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Calcium Binding to Calmodulin Mutants Having Domain-Specific Effects on the Regulation of Ion Channels

机译：钙结合钙调蛋白突变体对离子通道的调节具有特定域影响

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Calmodulin (CaM) is an essential,eukaryotic protein comprised of two highly homologous domains (N and C).CaM binds four calcium ions cooperatively,regulating a wide array of target proteins.A genetic screen of Paramecia by Kung [Kung,C.et al.(1992) Cell Calcium 13,413-425] demonstrated that the domains of CaM have separable physiological roles:"under-reactive" mutations affecting calcium-dependent sodium currents mapped to the N-domain,while "over-reactive" mutations affecting calcium-dependent potassium currents localized to the C-domain of CaM.To determine whether and how these mutations affected intrinsic calcium-binding properties of CaM domains,phenylalanine fluorescence was used to monitor calcium binding to sites I and II (N-domain) and tyrosine fluorescence was used to monitor sites III and IV (C-domain).To explore interdomain interactions,binding properties of each full-length mutant were compared to those of its corresponding domain fragments.The calcium-binding properties of six under-reactive mutants (V35I/D50N,G40E,G40E/D50N,D50G,E54K,and G59S) and one over-reactive mutant (M145V) were indistinguishable from those of wild-type CaM,despite their deleterious physiological effects on ion-channel regulation.Four over-reactive mutants (D95G,S101F,E104K,and H135R) significantly decreased the calcium affinity of the C-domain.Of these,one (E104K) also increased the calcium affinity of the N-domain,demonstrating that the magnitude and direction of wild-type interdomain coupling had been perturbed.This suggests that,while some of these mutations alter calcium-binding directly,others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel.
机译：钙调蛋白（CaM）是必不可少的真核蛋白，由两个高度同源的结构域（N和C）组成。CaM协同结合四个钙离子，调节广泛的靶蛋白。等人（1992）Cell Calcium 13,413-425]证明CaM的结构域具有可分离的生理作用：“低反应”突变影响映射到N结构域的钙依赖性钠电流，而“高反应”突变影响钙依赖的钾电流位于CaM的C结构域。为了确定这些突变是否以及如何影响CaM结构域的内在钙结合特性，使用苯丙氨酸荧光监测钙与I和II位（N结构域）和酪氨酸的结合荧光用于监测III和IV位点（C-结构域）。为探索域间相互作用，将每个全长突变体的结合特性与其相应的结构域片段的结合特性进行了比较。六个反应不足的突变体（V35I / D50N，G40E，G40E / D50N，D50G，E54K和G59S）和一个反应过度的突变体（M145V）与野生型CaM突变体没有区别，尽管它们对离子四个过度反应的突变体（D95G，S101F，E104K和H135R）显着降低了C结构域的钙亲和力。其中一个（E104K）也增加了N结构域的钙亲和力，表明野生型域间偶联的强度和方向受到干扰。这表明，尽管其中一些突变直接改变了钙结合，但其他可能改变了CaM通道缔合或钙触发的构象变化。受影响的离子通道。

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